Late Pregnancy is a Critical Period for Changes in Phosphorylated Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase 1/2 in Oxytocin Neurones.

The physiological demands of parturition and lactation lead to the increased pulsatile release of oxytocin (OT) into the circulation from the neurohypophysial axons of OT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. These states of increased OT release are accompanied by a significant plasticity in magnocellular OT neurones and their synaptic connections, and many of these changes require activation of a central OT receptor. The mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK) is assumed to be up-regulated in the PVN during lactation, and many of the effects of OT in peripheral and brain tissue are mediated through a MAPK/ERK pathway. The present study investigated whether this pathway is altered in the SON and PVN during late pregnancy [embryonic day (E)20-21], which is a critical period for OT plasticity induction, and for lactation, when plastic changes are sustained. Based on immunoreactivity for phosphorylated ERK1/2 (pERK1/2), the results suggest an enhanced activation of MAPK/ERK pathway in OT neurones specifically during late pregnancy in both the SON and PVN. Although immunoblots from the SON confirm this pregnancy-associated up-regulation in late pregnancy, they also suggest enhancement into lactation as well. Together, the results suggest an important role for the MAPK/ERK pathway during reproductive changes in the SON and PVN.

The D2s dopamine receptor stimulates phospholipase D activity: a novel signaling pathway for dopamine.

The D2 dopamine receptor isoforms signal to a variety of cellular effectors in both the central nervous system and periphery. Two alternative splice forms of the D2 dopamine receptor exist, the D2s (short) and D2l (long), which has an insertion of 29 amino acids in the third intracellular loop (). In cells of the anterior lobe of the pituitary, D2 dopamine receptors (both forms) are present on lactotroph cells coupled to the inhibition of adenylyl cyclase, activation of voltage-gated calcium channels, and inhibition of potassium channels. We describe here a novel signaling pathway for the D2s, which is the activation of phospholipase D (PLD). GH4C1 cells, a clonal line derived from a rat pituitary tumor, were stably transfected with the gene encoding the D2s, generating GH4-121 cells. Treatment of GH4-121 cells with a dopaminergic agonist resulted in activation of PLD in both a dose-dependent and time-dependent manner. This signaling pathway was not inhibited by prior treatment of cells with pertussis toxin at concentrations that ablate other D2s receptor signaling in this cell line. The stimulation of PLD activity by D2s appeared to correlate with the presence of a specific protein kinase C isoform, PKCepsilon. The D2s stimulation of PLD activity was blocked by preincubation of cells with C3 exoenzyme, indicating that the stimulation of PLD may involve Rho family members. The stimulation of PLD by dopaminergic agonists took place in the absence of any detectable stimulation of phosphoinositide metabolism.

Atypical SCH23390 binding sites are present on bovine adrenal medullary membranes.

D1-selective dopamine receptor agonists inhibit secretagogue-stimulated catecholamine secretion from bovine adrenal chromaffin cells. The purpose of the studies reported here was to use the radiolabeled D1-selective dopamine receptor antagonist, SCH23390, to characterize putative D1-like dopamine receptors responsible for this effect. Characterization of SCH23390 binding sites demonstrated an unusual pharmacological profile inconsistent with classical D1-like receptors. [125I]SCH23390 bound to adrenal medullary membranes was competed for by nonradioactive iodo-SCH23390 (Kd = 490 +/- 50 nM), but not by (+)butaclamol. Other classical D1 antagonists had little, if any, effect. Competition with dopamine receptor agonists demonstrated a relative rank order of potency profile characteristic of D1-like dopamine receptors, however, K(i)s were higher than those found in other tissues. The K(i)s for competition of [125I]SCH23390 binding by Cl-APB and SKF38393 (16 and 118 microM, respectively) are nearly identical to the IC(50)s previously observed for inhibition of secretion (9 and 100 microM, respectively). Combined these data suggest that adrenal medullary membranes contain a novel SCH23390 binding site involved in the inhibition of secretion by D1-selective agonists.

Differential inhibition of secretagogue-stimulated sodium uptake in adrenal chromaffin cells by activation of D4 and D5 dopamine receptors.

Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca2+ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na+, which could explain inhibition of secretion and Ca2+ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated 22Na+ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 microM) and SKF-38393 (< 00 microM) inhibited DMPP-stimulated Na+ uptake by 87.5 +/- 2.3 and 59.7 +/- 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 microM) inhibited Na+ uptake by only 22.9 +/- 5.0%. Veratridine-stimulated Na+ uptake was inhibited 95.1 +/- 3.2 and 25.7 +/- 4.7% by 100 microM CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC50 (approximately 18 microM) for inhibition of both DMPP- and veratridine-stimulated Na+ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 mM) had no effect on either DMPP- or veratridine-stimulated Na+ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na+ uptake in a cyclic AMP-independent manner.

Dopaminergic inhibition of catecholamine secretion from chromaffin cells: evidence that inhibition is mediated by D4 and D5 dopamine receptors.

Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K+ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzazepine (CI-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with approximately 10 microM CI-APB and approximately 100 microM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by CI-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca2+ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The D2 dopamine receptor isoforms signal through distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with site-directed mutant Gi alpha proteins.

Site-directed mutations of the cDNA for Gi1 alpha, Gi21 alpha, and Gi3 alpha were constructed which changed the cysteine residue at the C terminus to a glycine residue (Gi alpha PT). This mutation of the Gi alpha would not permit the subsequent covalent modification by pertussis toxin, which requires the cysteine moiety. The cDNA for each of the mutant Gi alpha subunits was transfected into GH4C1 cells with either of the alternative splice forms of the D2 dopamine receptor and clonal lines were generated. After treatment with pertussis toxin to remove the contribution from endogenous Gi proteins, the receptor-mediated inhibition of adenylyl cyclase was examined. The D2 dopamine receptor, short form (D2s) signaled through the Gi2 alpha PT mutant in these cells with an affinity for agonist which was comparable to that observed in cells transfected with the cDNA for D2s alone or the signaling observed in the absence of pertussis toxin. The long form of the D2 dopamine receptor (D2l) signaled through the Gi3 alpha PT mutant to inhibit forskolin-stimulated adenylyl cyclase, with an affinity for agonist comparable to that observed in cells transfected with the cDNA for D2l alone. The receptor for somatostatin (somatotropin release inhibiting factor) was used as an endogenous control receptor in these cell lines. The somatotropin release inhibiting factor was able to signal through both Gi1 alpha PT and Gi3 alpha PT to inhibit forskolin-stimulated adenylyl cyclase. These results indicated that receptors use distinct Gi proteins to signal to a common effector.

The D2 dopamine receptor mediates inhibition of growth in GH4ZR7 cells: involvement of protein kinase-C epsilon.

The D2 dopamine agonist, bromocriptine, has been used as treatment for human PRL-secreting pituitary adenomas. The result of bromocriptine treatment is often a substantial reduction of tumor mass, suggesting that the dopamine agonist is acting as an antiproliferative agent. This action can be observed with a clonal pituitary tumor cell line. The agonist activation of the D2 dopamine receptor inhibits the growth of GH4ZR7 cells, a GH4C1 cell line stably transfected with the cDNA encoding the short form of the D2 dopamine receptor. This effect of dopamine was not sensitive to overnight treatment with 100 ng/ml pertussis toxin. Treatment of GH4ZR7 cells with the phorbol ester 4 beta-phorbol 12,13-didecanoate resulted in the loss of dopaminergic inhibition of growth, whereas treatment with 4 alpha-phorbol 12,13-didecanoate had no effect. Inhibitors of protein kinase-C (PKC), such as staurosporine and H7, also blocked the effect of dopamine. Down-regulation of cellular PKC by phorbol ester treatment resulted in a complete loss of dopaminergic inhibition of growth. Long term treatment of GH4ZR7 cells with TRH results in a specific down-regulation of the epsilon form of PKC and abolished the ability of dopamine to inhibit growth. These results suggest that PKC epsilon is involved in mediating the antiproliferative effects of dopamine. This mediation of growth appears to be through a novel signaling pathway for the D2 dopamine receptor.

Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization.

The regulation of cellular responsiveness to dopamine via the D2 dopamine receptor was investigated in mouse fibroblast Ltk-cells stably expressing the rat D2-short receptor [Nature (Lond.) 336:783-787 (1988)]. Dopamine inhibited forskolin-stimulated cAMP levels in these cells (half-maximal inhibition at 3.9 +/- 1.1 nM), and the inhibition by dopamine was blocked by D2 antagonists and was pertussis toxin sensitive. Treatment of these cells with the D2 agonist quinpirole (1 microM) resulted in desensitization of dopaminergic inhibition of forskolin-stimulated cAMP accumulation, with a approximately 4-fold decrease in the potency of dopamine after 1 hr of treatment. No significant changes in total cellular D2 receptor concentrations were observed, even after prolonged agonist treatment. At longer time points, basal and forskolin-stimulated cellular cAMP levels were increased in treated cells. The effect of D2 agonist treatment on membrane adenylyl cyclase (EC 4.6.1.1) activity was examined. Basal and forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were increased by quinpirole treatment for 24 hr. This sensitization of adenylyl cyclase was blocked by the presence of a D2 antagonist. Pertussis toxin pretreatment blocked the sensitization of adenylyl cyclase by quinpirole, although pertussis toxin also caused increased adenylyl cyclase activity on its own. Sensitization was not dependent upon dopaminergic inhibition of intracellular cAMP levels, because quinpirole treatment in the presence of membrane-permeable cAMP analogs or 3-isobutyl-1-methylxanthine (an inhibitor of cAMP phosphodiesterase) resulted in greater sensitization of adenylyl cyclase activity than quinpirole treatment alone. These results suggest that, in this model system, responsiveness to dopamine via the D2 receptor is regulated by both desensitization of receptor function and sensitization of the stimulatory adenylyl cyclase pathway.

Molecular characterization of dopamine receptors.

The D1 and D2 dopamine receptors have been biochemically characterized using specific probes based on the subtype selective antagonists SCH 23390 and spiperone, respectively. The D2 dopamine receptor was identified from several tissues by photoaffinity labeling and was purified from bovine anterior pituitary to homogeneity using a combination of affinity, lectin and hydroxylapatite chromatography. A complementary DNA (cDNA) encoding a rat brain D2 dopamine receptor has been cloned via low stringency hybridization using a portion of the beta 2-adrenergic receptor gene as a probe. Photoaffinity crosslinking and affinity chromatography have also been used to identify and purify the rat brain D1 dopamine receptor.

Dopamine receptor subtypes: beyond the D1/D2 classification.

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system.

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe.

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.

Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary.

The D2-dopamine receptor from bovine anterior pituitary has been purified approximately 33,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized carboxymethyleneoximinospiperone-Sepharose, Datura stramonium lectin-agarose, and hydroxylapatite chromatography. The purification yields a single polypeptide band of Mr approximately 120,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed by labeling with radioiodinated Bolton-Hunter reagent, Coomassie Blue, or silver staining. The purified D2 receptor preparations display a specific activity of approximately 5.3 nmol of [3H]spiperone bound per mg of protein. In detergent solutions, the purified receptor has a KD for [3H]spiperone of 5-8 nM; however, after reinsertion of the purified protein into phospholipid vesicles, a KD of approximately 160 pM is obtained, similar to that found for the receptor in crude membrane preparations. Several lines of evidence document that this polypeptide contains the ligand binding site as well as the functional activity of the D2 receptor. The Mr approximately 120,000 peptide can be covalently labeled by the affinity probe, 125I-bromoacetyl-N-(p-aminophenethyl)spiperone, with the pharmacological specificity expected of a D2-dopamine receptor. Agonist and antagonist ligands compete for [3H]spiperone binding to purified receptors in phospholipid vesicles with a rank order of potency and selectivity typical of a D2-dopamine receptor. Moreover, when reinserted into phospholipid vesicles with purified brain Gi/Go, the purified D2 receptors mediate the agonist stimulation of 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-proteins with a typical D2-dopaminergic order of potency. These data suggest that we have purified an intact functional D2-dopamine receptor.

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum.

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.

Biochemical properties of D1 and D2 dopamine receptors.

The physiological action of dopamine are mediated by two distinct subtypes of receptors, D1 and D2 dopamine receptors. D1-receptors are linked to stimulation of adenylate cyclase whereas D2-receptors inhibit the enzyme and may also couple to other signal transduction systems such as ion channels. In order to characterize these receptors at the biochemical level we have developed specific probes for the identification and purification of these proteins. The ligand binding sites of the two receptors have been identified by photoaffinity labeling and reside on distinct polypeptides. In rat striatum, the D1 receptor binding site can be identified as a peptide of Mr = 72,000. In contrast, the D2 receptors appears to reside on an Mr = 94,000 peptide in most tissues. A larger peptide of Mr = 120,000 identified in the intermediate lobe of pituitary may represent the unproteolyzed form of this receptor. An affinity chromatography purification procedure has been developed for the D2 dopamine receptor. This procedure affords a substantial purification (greater than 1000 fold) of the receptor solubilized from bovine anterior pituitary glands with complete retention of its binding properties. These biochemical tools should eventually lead to the complete characterization of these two receptor subtypes.

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein.

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.

Affinity chromatography of the anterior pituitary D2-dopamine receptor.

The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified approximately 1000-fold by affinity chromatography on a new affinity support. This support consists of a (carboxymethylene)oximino derivative of the D2-selective antagonist spiperone (CMOS) covalently attached to Sepharose 4B through a long side chain. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Dopaminergic drugs blocked adsorption of solubilized receptor activity to the CMOS-Sepharose with the appropriate D2-dopaminergic potency and stereoselectivity. For agonists, (-)-N-n-propylnorapomorphine greater than 2-amino-6,7-dihydroxytetrahydronaphthalene approximately equal to apomorphine greater than dopamine, whereas for antagonists (+)-butaclamol much greater than (-)-butaclamol. The same D2-dopaminergic specificity was observed for elution of receptor activity from the gel. To observe eluted receptor binding activity, reconstitution of the eluted material into phospholipid vesicles was necessary. Typically, 70-80% of the solubilized receptor was adsorbed by CMOS-Sepharose, and 40-50% of the adsorbed activity could be recovered after reconstitution of the eluted material. The overall recovery of D2-receptor activity from bovine anterior pituitary membranes was 12-15% with specific binding activity of approximately 150 pmol/mg. The reconstituted affinity-purified receptor bound ligands with the expected D2-dopaminergic specificity, stereoselectivity, and rank order of potency.

von Willebrand factor. A protein which binds at the cell surface interface between platelets.

von Willebrand factor (VWF) functions in platelet aggregation, a form of cellular interaction. In vitro analysis of platelet aggregation, as measured by the platelet aggregometer, requires addition of a promoter such as the glycopeptide antibiotic ristocetin. Native multimeric VWF (Mr = 1-20 X 10(6)) can be reduced with sulfhydryl reagents to a monomeric state (Mr = 2 X 10(5)). In this study, the binding of bovine VWF and ristocetin to bovine platelets was investigated using fluorescence anisotropy of derivatized monomer protein and ristocetin and also by radioisotope methods using 125I-labeled monomer and native protein. Ristocetin bound to bovine platelets but not to VWF. VWF binding to formaldehyde-fixed platelets was dependent on the presence of a promoter such as ristocetin. The monomer and multimer VWF bound equally well in the presence of low ristocetin concentrations. Under these conditions, plots of VWF binding versus platelet concentration were sigmoidal, indicating positive cooperativity with respect to platelets. At higher (100 micrograms/ml) ristocetin concentrations, the binding curve was no longer sigmoidal. Ristocetin promoted the formation of small platelet aggregates, an effect that was amplified by the presence of VWF. In fact, all conditions which resulted in monomer or multimer VWF binding to platelets also caused formation of platelet aggregates observed by light microscopy. These combined results were consistent with VWF binding only to the interface between proximal platelets. High affinity binding could be provided by the presence of two cell surfaces and the resulting multiple binding interactions. Polycations, such as poly(L-lysine) and Polybrene, also promoted the formation of platelet aggregates and facilitated the binding of VWF to platelets. Physiological platelet activators such as thrombin, ADP, and collagen also facilitated VWF binding to native platelets and caused platelet aggregation. It appears possible that any process which causes the surface membranes of platelets to become spatially close will allow expression of VWF activity.

A cholinergic role in the mechanism of lithium in mania.

Based on evidence that mania may include an alteration of cholinergic function, we have previously investigated the effects of various monovalent ions on the muscarinic cholinergic receptor from human caudate nucleus utilizing the radio-labeled antagonist (3H)quinuclidinyl benzilate (QNB). In this study we observed that Li+ at 1 mM was unique in its ability to specifically reduce the affinity of 3H-QNB for the muscarinic receptor (increasing the QNB dissociation constant from 35.9 pM to 72.4 pM). The sodium-specific induction of positive cooperativity (nHill) at the muscarinic cholinergic receptor was inhibited in the presence of Li+. Both observations achieved statistical significance at p less than 0.05. A Li+-related decrease in specific QNB binding sites (Bmax) by 23% (from 2480 to 1900 pmole/g protein) was additionally manifest, albeit only achieving a statistical trend (p less than 0.10). The exact mode of Li+ action in the management of major affective illnesses remains speculative. The observations that this cation specifically mediates a reduction of muscarinic receptor affinity and number of binding sites within the human caudate suggests further consideration of the association between muscarinic cholinergic activity and mania as it relates to Li+.