Peripheral dopamine receptor identification: Properties of a specific dopamine receptor in the rat adrenal zona glomerulosa

Specific dopaminergic receptors were found in the rat adrenal zona glomerulosa. Specific binding as defined by the difference in [³H]-spiroperidol binding in the presence or absence of excess dopamine was saturable and of high affinity. Stereospecificity of binding to the dopaminergic receptor was demonstrated by the fact that (+)-butaclamol was 300-fold more active at displacing [³H]-spiroperidol from the binding site than (?)-butaclamol. A Scatchard analysis of the data revealed a K_D = 6.9 nM and a B_max = 173 pmol/gm for the binding of [³H]-spiroperidol to adrenal capsular homogenate binding site. Characteristics of this receptor place it in the recently defined D2 dopamine receptor subclass.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

Purification of bovine striatal dopamine D-2 receptor by affinity chromatography

Bovine striatal dopamine D-2 receptor has been purified approximately 2000-fold by affinity chromatography. The receptor, solubilized with cholic acid and sodium chloride, was adsorbed on haloperidol-linked Sepharose CL-6B and eluted with spiroperidol. The adsorption of receptor to the affinity matrix was biospecific as preincubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor. The process also displayed stereoselectivity with respect to (+)- and (-)-butaclamol. Nondopaminergic agents such as mianserin and propranolol failed to exhibit any effect on the adsorption process. Elution of the receptor was also biospecific, as dopaminergic drugs were most effective (spiroperidol greater than haloperidol greater than dopamine) in eluting the bound receptor; whereas other agents, e.g. propranolol, mianserin, and acetic acid, were only slightly effective. One-cycle affinity purification resulted in a recovery of 12% of the original membrane-bound dopamine D-2 receptor with a specific activity of 169,600 fmol/mg of protein as assayed with [3H]spiroperidol binding. The order of potency of D-2 agonists (N-propylnorapomorphine greater than NO434 greater than apomorphine greater than dopamine) and antagonists (spiroperidol greater than (+)-butaclamol greater than domperidone) with the purified preparation was found to be similar to that of the solubilized dopamine D-2 receptor.     

Photoaffinity labeling of the D2-dopamine receptor using a novel high affinity radioiodinated probe

The ligand binding subunit of the D2 subtype of the dopamine receptor has been identified by photoaffinity labeling. In order to develop a specific covalent receptor probe, an analogue of the potent D2 selective antagonist spiperone, N-(p-aminophenethyl)spiperone (NAPS) has been synthesized. The aminophenethyl substituent of NAPS can be radioiodinated to theoretical specific radioactivity (2,175 Ci/mmol) and then the arylamine group converted to an arylazide to yield a photosensitive probe [( 125I]N3-NAPS). In rat striatal membranes, the nonradiolabeled azide probe (N3-NAPS) binds to the receptor with high affinity (KD congruent to 1.6 +/- 0.05 nM) and upon photoactivation irreversibly decreases the number of available receptors in these membranes as measured by [3H]spiperone binding. More importantly, however, incubation of rat striatal membranes with [125I]N3-NAPS leads to the photodependent covalent incorporation of the probe into a peptide of Mr = 94,000 as assessed by autoradiography of gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of this Mr = 94,000 peptide can be blocked specifically and stereoselectively by dopaminergic antagonists such as (+)- and (-)-butaclamol but not by non-dopaminergic antagonists. Moreover, dopaminergic agonists also attenuate the covalent labeling of this peptide with an order of potency which is typically D2-dopaminergic. Therefore, the specificity of [125I]N3-NAPS labeling of the Mr = 94,000 peptide suggests that this peptide represents the ligand binding subunit of the D2-dopamine receptor.     

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.     

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.     

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (?)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [³H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c-Jun mutant, also prevented stimulation of [³H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

The effects of dopamine receptor agonists and antagonists on the secretory rate of cockroach (Periplaneta americana) salivary glands

The acinar salivary glands of the cockroach, Periplaneta americana, are innervated by dopaminergic and serotonergic nerve fibers. Serotonin stimulates the secretion of protein-rich saliva, whereas dopamine causes the production of protein-free saliva. This suggests that dopamine acts selectively on ion-transporting peripheral cells within the acini and the duct cells, and that serotonin acts on the protein-producing central cells of the acini. We have investigated the pharmacology of the dopamine-induced secretory activity of the salivary gland of Periplaneta americana by testing several dopamine receptor agonists and antagonists. The effects of dopamine can be mimicked by the non-selective dopamine receptor agonist 6,7-ADTN and, less effectively, by the vertebrate D1 receptor-selective agonist chloro-APB. The vertebrate D1 receptor-selective agonist SKF 38393 and vertebrate D2 receptor-selective agonist R(-)-TNPA were ineffective. R(+)-Lisuride induces a secretory response with a slower onset and a lower maximal response compared with dopamine-induced secretion. However, lisuride-stimulated glands continue secreting saliva, even after lisuride-washout. Dopamine-induced secretions can be blocked by the vertebrate dopamine receptor antagonists cis(Z)-flupenthixol, chlorpromazine, and S(+)-butaclamol. Our pharmacological data do not unequivocally indicate whether the dopamine receptors on the Periplaneta salivary glands belong to the D1 or D2 subfamily of dopamine receptors, but we can confirm that the pharmacology of invertebrate dopamine receptors is remarkably different from that of their vertebrate counterparts.     

Monoclonal antibodies directed against the drug haloperidol

Haloperidol coupled to albumin via the hydroxyl or carbonyl group has been used to obtain two classes of monoclonal anti-haloperidol antibodies. Both classes of monoclonal antibody bind haloperidol with high affinity but whereas one class is highly specific for haloperidol the other class binds other butyrophenones e.g. droperidol, spiperone. Anti-idiotypic antisera have been obtained that inhibit [³H]haloperidol binding to the anti-haloperidol antibodies but these do not cross react with the D₂ dopamine receptor.     

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Pre- and postsynaptic effects of dopamine antagonists on dopaminergic synaptic transmission in Helix pomatia

The rate of transmitter mobilization in identified dopaminergic synapses was decreased by the dopamine antagonists pimozide, chlorpromazine, haloperidol, cis-flupenthixol, curare, clozapine and high concentrations of ergometrine. The depolarizing postsynaptic potential was inhibited by pimozide, chlorpromazine, haloperidol, cis-flupenthixol, curare, clozapine, (+)-butaclamol and high concentrations of ergometrine. The hyperpolarizing synaptic potential was inhibited by naloxone, methysergide, (+)-butaclamol, haloperidol, 6-hydroxydopamine and low concentrations of ergometrine, while pimozide, cis-flupenthixol, trans-flupenthixol, curare, clozapine, promethazine, chlorpromazine and (-)-butaclamol had no clear effect. The presynaptic receptors involved in modulation of the mobilization rate showed similarities with the dopamine receptors mediating depolarizations. The dopamine antagonists changed dynamics of synaptic transmission.     

Dopamine D2 receptors retain agonist high-affinity form and guanine nucleotide sensitivity after removal of sialic acid

The ligand binding subunit of the D2 dopamine receptor (Mr approximately equal to 94,000) can be visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr = 54,000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P]ADP ribosylation of proteins of Mr = 42,000 and 39,000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit.     

Dopamine D2 Receptors in the Anterior Pituitary: A Single Population Without Reciprocal Antagonist/Agonist States

Although dopamine agonists can recognize two states of the D2 dopamine receptor in the anterior pituitary (D2high and D2low), we examined whether the dopamine antagonists such as [3H]spiperone could recognize these two sites with different affinities. Using up to 30 concentrations of [3H]spiperone, however, we could only detect a single population of binding sites (porcine anterior pituitary homogenates) with a dissociation constant (KD) of 130 pM. When specific [3H]spiperone binding was defined by a low concentration of (+)-butaclamol (100 nM), the apparent density was low. When defined by a high concentration of (+)-butaclamol (10 microM), nonspecific sites became detectable, thus revealing two apparent populations of sites for [3H]spiperone, only one of which was specific for dopamine. Sodium chloride reduced the KD of the single population of specific D2 sites to 64 pM. Guanine nucleotide by itself had no effect on the KD, but enhanced the density by 25%. Since the density-enhancement could be eliminated by extensive washing of membranes, and could be restored by preincubation with dopamine, the nucleotide-induced elevation of D2 density appeared to be a result of the release of tightly bound endogenous dopamine. Thus, monovalent cations and guanine nucleotides appear to have separate regulatory effects on the anterior pituitary D2 receptor that modulate antagonist-receptor interactions. Several maneuvers were used to test whether [3H]spiperone could differentiate between the two agonist-detected subpopulations of sites. Twentyfold different concentrations of [3H]spiperone (47 pM and 1000 pM) were found to label identical proportions of receptors in the D2high and D2low states as detected by the agonist 6,7-dihydroxyaminotetralin (ADTN), suggesting that spiperone labelled equal proportions of D2high and D2low sites without differential affinity for them. In addition, competition of spiperone for D2high sites selectively labelled by the agonist [3H]n-propylnorapomorphine (NPA) had a virtually identical KD for spiperone as did the total D2 receptor population as determined by direct binding studies (75 pM versus 64 pM). [3H]Spiperone also bound to a uniform population of D2low sites induced by preincubation with guanine nucleotide with identical affinity as to the total D2 population. Thus, these data do not support a "reciprocal model" for the D2 receptor (i.e., antagonist having low affinity for D2high and high affinity for D2low in a manner reciprocal to agonists).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Dietary n-3 Fatty Acids on the Phospholipid Molecular Species of Monkey Brain

Y-79 human retinoblastoma cells grown in serum-free medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y-79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y-79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of approximately 3 fmol/10(6) cells (approximately 1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly-D-lysine and fibronectin and continued culture in serum-free medium. After 8 days in serum-free culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/10(6) cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15-17 days (7-9 days of Bt2cAMP treatment) expressing receptor levels of 15-20 fmol/10(6) cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y-79 (dY-79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM-09151-2 approximately spiperone greater than domperidone approximately (+)-butaclamol approximately fluphenazine greater than chlorpromazine greater than (-)-sulpiride greater than (+)-sulpiride greater than promethazine greater than (+)-SCH 23390 much greater than (-)-butaclamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Binding of 3H-lisuride hydrogen maleate to striatal membranes of rat brain

Binding of ³H-lisuride hydrogen maleate (LHM), a dopaminergic agonist, to striatal membranes was inhibited by (+)-butaclamol, whereas (-)- butaclamol at a concentration of 10^?9-10^?7M was without effect. The difference in the amount of ³H-LHM bound to striatal membranes in the presence of 10^?7 M (-)-butaclamol and (+)-butaclamol was designated as the specific binding of ³H-LHM, and its properties were examined. The specific ³H-LHM binding to striatal membranes was saturated with an equilibrium amount of 490 fmol/mg protein and had an apparent dissociation constant (Kd) of 0.5 nM. The specific binding of ³H-LHM to striatal membranes was inhibited by LHM, haloperidol, apomorphine and methysergide with inhibitor association constants (Ki value) of 0.79, 7.1, 100 and 180 nM, respectively. Phentolamine, dopamine, (-)-norepinephrine and serotonin were weaker inhibitors of the specific binding of ³H-LHM to striatal membranes, with Ki values of 1,100, 3,500, 33,000 and 79,000 nM, respectively. No inhibition was observed with (±)-propranolol, dichloroisoproterenol or QNB. These results are discussed in connection with dopamine receptors.     

Specific binding of 3H-SCH 23390 to dopamine D1 receptors in vivo

The binding of 3H-SCH 23390 was studied in vivo in the mouse brain. The binding was saturable, reversible and stereospecific. The level of nonspecific binding was 5-15% of total binding. Pharmacological characterization revealed binding of 3H-SCH 23390 to D1 receptors. Thus, dopaminergic antagonists known to possess D1 affinity, e.g., SCH 23390 itself, cis-flupentixol and (+)-butaclamol, were potent inhibitors of the 3H-SCH 23390 binding. On the other hand, high doses of D2 selective compounds were required to inhibit the 3H-SCH 23390 binding. These results indicate that 3H-SCH 23390 is a ligand of choice for in vivo studies of D1 receptors.     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.