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PubMed1981

Lack of postulated opiate-receptor antagonistic properties of compound 48/80.

The previously suggested opiate-receptor antagonistic properties of compound 48/80 were checked both in vivo and in vitro. In electrically stimulated guinea pig ileum and rat vas deferens preparations compound 48/80 did not reverse the inhibition of the twitches caused either by morphine or Met5-enkephalin. In mice hot-plate test compound 48/80 did not decrease the analgesic activity of morphine. In radioreceptor studies compound 48/80 shows rather low affinity to the 3H-naloxone receptor sites from striatal homogenates of the rat, as compared to non-labelled naloxone and on the other hand it produced moderate, as compared to d-butaclamol, displacement of 3H-spiroperidol from rat striatal homogenates. The variation of composition of various polymers making compound 48/80 is suggested for explanation of obtained results.

Analgesics↗
PubMed1984

Two types of calmodulin antagonists: a structurally related interaction.

The ability of several calmodulin (CaM) antagonists, such as N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine, to displace [3H]-W-7 from CaM correlated with the inhibition of Ca2+-CaM-dependent phosphodiesterase (PDE) by these agents. These antagonists also suppressed the increase in fluorescence of n-phenyl-1-naphthylamine (NPN) by complex formation with CaM in the presence of Ca2+. However, the ability of some CaM antagonists, such as prenylamine and butaclamol, to displace [3H]-W-7 from CaM did not correlate with the inhibition of Ca2+-PDE. These antagonists enhanced the increase in fluorescence of NPN by complex formation with CaM in the presence of Ca2+. In a study employing 1H-nuclear magnetic resonance, the spectrum changes of the aromatic region of CaM induced by prenylamine were significantly more marked than the changes induced by W-7. These findings suggest two types of CaM antagonists. The compounds in each of the groups appear to have common molecular structures.

Animals↗
PubMed1984

Interaction of sulpiride with human pituitary dopaminergic receptors is sodium dependent.

The effects of various drugs on [3H]-spiroperidol binding to human anterior pituitary and prolactinoma membranes are studied in the presence and in the absence of sodium chloride in the incubation medium. It is shown that in the absence of NaCl, 1-sulpiride is significantly less potent in displacing the radioactive ligand from its dopaminergic binding sites than in the presence of NaCl. On the contrary, the interaction of neuroleptics (d-butaclamol and haloperidol) and dopamine agonists (apomorphine and bromocriptine) was unaffected by the NaCl conditions.

Binding, Competitive↗
PubMed1986

Dopaminergic binding and inhibitory effect in the bovine adrenal zona glomerulosa.

Dopaminergic mechanisms may be involved in the regulation of aldosterone secretion in humans and in the rat. Whether these effects are indirect or are exerted directly at the adrenal level has not yet been resolved. We now report the identification of dopaminergic binding sites in the bovine adrenal zone glomerulosa using [3H]spiperone, a butyrophenone with high affinity for D2 dopamine receptors. Specific [3H]spiperone binding (defined as binding displaceable by 10 microns (+)-butaclamol) reached equilibrium within 20 minutes at 22 degrees C, was reversible, and was heat labile (60 degrees C). Binding was of high affinity and saturable with a Kd of 1.8 +/- 0.2 nM and maximal specific binding of 38 +/- 8 fmol/mg (means +/- SEM; n = 18). [3H]Spiperone binding was unaffected by coincubation with angiotensin II, adrenocorticotropic hormone, or KCl. Binding characteristics, including a dissociation constant at the nanomolar range, greater potency of the D2-agonist LY 171555 relative to the D1-agonist SKF 38393 in inhibiting [3H]spiperone binding, and lack of stimulation of cyclic adenosine 3',5'-monophosphate by dopamine (10(-4) M), were consistent with a predominantly D2-receptor. In vitro studies with collagenase-dispersed adrenal zona glomerulosa cells showed that dopamine (10(-4) M) attenuated angiotensin II-stimulated aldosterone secretion. These observations are consistent with a direct inhibitory effect of dopamine on aldosterone secretion in the adrenal zona glomerulosa.

Adrenal Cortex↗
PubMed1978

Effect of haloperidol and apomorphine treatment on dopamine receptors in pituitary and striatum.

Prompted by an interest in the similarity of brain and tuberoinfundibular systems, the authors studied butaclamol-specific neuroleptic and apomorphine binding in pituitary and striatum after chronic haloperidol and acute apomorphine treatment. Striatal binding increases but pituitary binding decreases in haloperidol-treated rats. Pituitary binding changes rapidly in response to apomorphine exposure and striatal binding does not. These findings suggest that factors influencing binding differ in these two tissues.

Animals↗
PubMed1979

Dopamine receptor sites in the anterior pituitary.

An immunocytochemical method was developed to visualize dopamine receptor sites on dispersed anterior pituitary cells of the rat. Dopamine receptors were labeled with the antagonist haloperidol. Some cells were incubated with haloperidol and a 100-fold excess of the potent antagonist D-butaclamol to determine nonspecific binding. The labeled sites were stained with an antibody against haloperidol and the peroxidase anti-peroxidase (PAP) technique. PAP complexes which served as markers for dopamine binding sites appeared on the outer plasmalemmal surface of the vast majority of mammotrophs. PAP complexes attached to the inner surface of endocytotic vesicle membrane suggested internalization of receptor-rich portions of the plasmalemma. Some gonadotrophs and somatotrophs were specifically stained to a lesser extent. However, high receptor site density and internalization of PAP complexes were never observed on cell types other than mammotrophs. The presence of dopamine receptors on the plasmalemma of mammotrophs provides strong additional evidence that dopamine acts upon these cells as a prolactin inhibitory hormone.

Animals↗
PubMed1984

Ovarian steroids modulate the release of dopamine into hypophysial portal blood and the density of anterior pituitary [3H]spiperone-binding sites in ovariectomized rats.

The concentrations of dopamine (DA) in pituitary stalk plasma and the number of DA D-2 receptor sites in the anterior pituitary gland were evaluated in ovariectomized (OVX) rats treated for 48-78 h with estradiol (E2) or vehicle and for 2-30 h with progesterone (P) or vehicle. Only the combined administration of E2 for 72-78 h and P for 24-30 h altered the release of DA into pituitary stalk blood and the number of dopaminergic binding sites in the anterior pituitary gland. The DA concentrations in pituitary stalk plasma 72 h after initiation of treatment were 1.9 +/- 0.3 (mean +/- SEM), 2.6 +/- 0.2, and 2.4 +/- 0.3 ng/ml in OVX rats treated with vehicle, E2, and P alone, respectively, and were increased significantly to 4.3 +/- 0.4 ng/ml in E2- and P-treated rats. The binding of [3H] spiperone to DA D-2 receptors was measured by a single point assay in homogenates of the individual anterior pituitary glands obtained from the same rats. Anterior pituitary tissue was incubated with 0.4 nM [3H]spiperone in the presence or absence of 10(-6) M (+)butaclamol. Binding was similar in all animals except those treated with E2 and P, in which a significant increase occurred. Three days after initiation of treatment, binding was equivalent to 49.8 +/- 5.8, 51.3 +/- 5.1, and 55.4 +/- 4.9 fmol/mg protein in OVX rats treated with vehicle, E2, and P, respectively. In contrast, a significant increase in binding to 83.7 +/- 4.6 fmol/mg protein was observed in rats given E2 and P. No differences in either parameter were apparent 48 h after starting the steroid/vehicle treatment, nor were there any differences between morning and afternoon values in any single group. Scatchard analyses were made on the binding kinetics of DA receptors in anterior pituitary glands obtained from additional rats, and 1.5- to 2-fold increases in the density of sites were observed in animals that had received both E2 and P, while the apparent affinity of the receptors was unchanged. We conclude that treatment with the combination of E2 and P is required to increase the release of DA into hypophysial portal blood and that it also increases the density of DA receptors within the anterior pituitary gland.

Animals↗
PubMed1988

Dopamine inhibits prolactin secretion stimulated by the calcium channel agonist Bay-K-8644 through a pertussis toxin-sensitive G protein in anterior pituitary cells.

In primary culture of anterior pituitary cells, BAY-K-8644, a calcium channel agonist, stimulated PRL secretion by 83% with EC50 of 18 nM. This effect was blocked by nifedipine, a calcium channel antagonist. The stimulations of PRL secretion induced by potassium (50 mM) and BAY-K-8644 were additive. Dopamine inhibited basal as well as BAY-K-8644-stimulated PRL secretion by 64% and 75%, respectively, and with respective EC50 values of 4.5 and 0.6 nM. In the presence of 50 mM K+, dopamine only partially blocks the dose-dependent stimulation of PRL secretion induced by the calcium channel agonist. The inhibitory dopamine effect was blocked by (+)butaclamol, a specific dopamine receptor antagonist. The dopamine response was also blocked by 1-sulpiride, a specific dopamine D2 receptor antagonist, and mimicked by RU 24926, a specific dopamine D2 receptor agonist, suggesting that the dopamine effect on BAY-K-8644-stimulated PRL secretion was mediated through a D2 dopamine receptor. Although unknown, the mechanism by which dopamine inhibited the BAY-K-8644-stimulated PRL secretion involves a GTP binding protein sensitive to Bordetella pertussis toxin. In fact, the dopamine inhibition of PRL secretion induced by the calcium channel agonist was blocked by the pretreatment of cells with the toxin. These results suggest that dopamine D2 receptors in lactotroph cells modulate calcium influx through a GTP binding protein.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1982

Evidence for the presence of D2 and 5-HT2 receptors in the human prefrontal cortex.

The D2 receptor and two distinct affinity 5-HT2 receptors in the human prefrontal cortex were labeled with 3H-spiperone. In the Scatchard analysis of stereoselective binding defined by (+)- and (-)-butaclamol, two high affinity binding sites were clearly demonstrated. Apparent KD values of these higher and lower affinity sites determined in this manner were 0.028 and 0.64 nM, respectively. However, drug displacement studies of 3H-spiperone binding at these sites, using different concentrations of 3H-spiperone (0.05 nM for higher affinity sites and 0.5 nM for lower affinity sites), indicated that 70-80% of the higher affinity sites and almost all of the lower affinity sites showed characteristics of the 5-HT2 receptor and that 20-30% of the higher affinity sites showed characteristics of the D2 dopamine receptor. An additional saturation experiment related to the specific binding of 3H-spiperone to 5-HT2 receptors confirmed the existence of two distinct populations of 5-HT2 receptors that had different affinities for spiperone. Apparent KD values for higher and lower affinity 5-HT2 receptors were 0.091 and 1.27 nM, respectively. As the D2 receptor is a possible site of antipsychotic action of neuroleptics, these receptors, especially the D2 receptor in the human prefrontal cortex, seem to play an important role in psychotic disorders.

Binding, Competitive↗
PubMed1987

Dopamine stimulates release of thyrotrophin-releasing hormone from perfused intact rat hypothalamus via hypothalamic D2-receptors.

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.

Animals↗
PubMed1997

Chimeric dopamine D2/angiotensin AT1 receptors: role of the length of third intracellular loop of D2 receptors in conferring specificity of receptor binding and G-protein coupling.

AIM: To define roles of the third intracellular loop (IL3) length of G-protein coupled receptors in conferring the specificity for receptor binding and G-protein coupling. METHODS: By polymerase chain reaction (PCR), the IL3 of D2 receptor was replaced with the counter part of AT1 receptor which has the shortest loop among all G-protein coupled receptors. D2/AT1 receptor cDNA was then stably transfected into Chinese hamster ovary cells and a clone with high level expression was obtained for receptor binding and agonist-induced phosphatidylinositols (PI) turnover experiments. RESULTS: Comparing to the D2 receptor, D2/AT1 chimeric receptor had lower affinities for all D2 receptor antagonists tested (spiperone, haloperidol, (+)-butaclamol, chlopromazine, clozapine, trifluoperdazine) and different affinity profiles to agonists (apomorphine, dopamine, quinpirole, bromocriptine). But the chimeric receptor failed to couple to G-protein and subsequent stimulation of PI turnover. CONCLUSION: The length of IL3 of D2 receptor participates defining recpetor binding sites conformation, and structure beyond IL3 may affect receptor G-protein coupling.

Animals↗
PubMed2001

Dynamic dopamine-antagonist interactions at recombinant human dopamine D(2short) receptor: dopamine-bound versus antagonist-bound receptor states.

Antipsychotic drugs comprise a wide range of structurally diverse compounds and are considered to be antagonists at dopamine D2 receptors. High-resolution kinetic analyses of their antagonist properties was performed by monitoring dynamic dopamine (DA)-antagonist interactions at the recombinant human dopamine D(2short) receptor. Time-dependent Ca2+ responses were measured following activation of a chimeric G(alphaq/o) protein in Chinese hamster ovary-K1 cells. DA (10 microM) induced a rapid, high-magnitude Ca2+ response (T(max) = 13.2 +/- 0.7 s) followed by a low-magnitude phase, which continued throughout the recorded time period (15 min). Of a large series of putative DA antagonists, (+)-UH 232 and bromerguride demonstrated positive, DA-like intrinsic activity at the presumably unoccupied, DA-free receptor; the other antagonists being silent. Antagonists differed in terms of their abilities to prevent the high-magnitude Ca2+ phase in the antagonist-bound receptor state, and to reverse the low-magnitude Ca2+ phase in the DA-bound state. The benzamide derivatives tropapride and nemonapride fully antagonized both the high- and low-magnitude Ca2+ response. Haloperidol, risperidone, and S 14066 also antagonized both responses but with a maximal effect of only 62 to 79%. Although preventing the high-magnitude response (85-95%), the further putative antagonists (+)-butaclamol (6%), bromerguride (27%), and domperidone (41%) reversed the low-magnitude response only weakly and partially. These Ca2+ data indicate that putative DA antagonists act differently, in particular, at the DA-bound D(2short) receptor.

Animals↗
PubMed1990

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Characterization and distribution of [125I]epidepride binding to dopamine D2 receptors in basal ganglia and cortex of human brain.

The distribution and pharmacology of the binding of [125I]epidepride, a substituted benzamide with high affinity and selectivity for dopamine (DA) D2 receptors in rat brain (Neve et al., J. Pharmacol. Exp. Ther. 252: 1108-1116, 1990), is described in human brain. Saturation analysis of the binding of [125I]epidepride to membranes derived from striatum and regions of cortex demonstrated similar Kd values (34 and 28-33 pM, respectively) but differing maximum density of binding site values (152 and 3-8 fmol/mg of protein, respectively). The pharmacological profile of binding in cortex was also similar to striatum (epidepride greater than spiperone greater than butaclamol = flupenthixol greater than clozapine) except that an additional low-affinity site, blocked by the alpha-2 adrenergic antagonist idazoxan, was present in cortex. Quantification by autoradiography also demonstrated the greatest binding in the basal ganglia, with the striatum exhibiting greater binding than the pallidal complex or midbrain regions. For the pallidum, binding in the external segment was higher than the internal segment. Within the midbrain the binding of [125I]epidepride correlated well with the known distribution of DA-containing cell bodies, with the substantia nigra (pars compacta and pars lateralis) and ventral tegmental area (A10) higher than area A8 and central gray. Binding in frontal and parietal cortex was highest in the internal layers (layers V and VI). Temporal cortex showed a 2-fold higher density of binding than other cortical regions and a trilaminar pattern; binding was greater in the external (layers I and II) and internal layers than in the middle layers (III and IV). This pattern changed in the parahippocampal complex. Within the lateral occipitotemporal cortex, binding was densest in layers I to III and very low in layers IV to VI, but binding was almost nonexistent in the adjacent entorhinal cortex. Within the hippocampal complex, binding was evident in the subiculum, CA3 and dentate gyrus and almost nonexistent in the presubiculum or other fields of the hippocampus. This pattern of binding in the parahippocampal gyrus is unique to human brain and represents sites of action for DA in limbic cortex.

Adult↗
PubMed1991

Behavioral effects of D1 and D2 dopamine receptor antagonists in squirrel monkeys.

The behavioral effects of dopamine antagonists differing in affinity and selectivity at D1 and D2 dopamine receptors were compared in squirrel monkeys responding under a fixed-interval schedule of stimulus-shock termination. D1-selective antagonists included (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7 -ol, SCH 23390; its enantiomer (S)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7 -ol, SCH 23388; [(-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H - benzo(d)naphtho-(2,1-b)azepine], SCH 39166; (R)-7-bromo-8-hydroxyl-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaze pine, R-SKF 83566; (R)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol, R-SKF 83692; 2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol, RS-SKF 83692. D2-selective antagonists included cis-N-(1-benzyl-2-methylpyrrolidine-3-yl)-5-chloro-2-methoxy-4- methylaminobenzamide, YM-09151-2, eticlopride, raclopride, haloperidol, risperidone, remoxipride, S-sulpiride and R-sulpiride; nonselective dopamine antagonists were S-butaclamol and chlorpromazine. Regardless of selectivity for D1 or D2 receptors, all drugs produced dose-related decreases in fixed-interval responding. A high degree of stereoselectivity was evident for both D1 antagonists (SCH 23390 and R-SKF 83692 more potent than, respectively, SCH 23388 and RS-SKF 83692) and D2 antagonists (S-sulpiride more potent than R-sulpiride). High doses of the D1 and D2 antagonists also reduced motor activity and impaired coordination in monkeys in the home cage after test sessions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Agonist-induced desensitization of D1-dopamine receptors linked to adenylyl cyclase activity in cultured NS20Y neuroblastoma cells.

NS20Y neuroblastoma cells expressing a homogeneous population of D1-dopamine receptors were used in the present study as a model system to investigate the mechanisms of agonist-induced stimulation and desensitization of D1 receptor-coupled adenylyl cyclase activity. Membrane prepared from NS20Y cells showed a pharmacologically specific, dose-dependent increase in cAMP production in response to various dopaminergic agonists. Dopamine exhibited an EC50 of 5 microM, and at 100 microM a maximal stimulation of 3-4-fold over basal enzyme activity was observed, which could be selectively antagonized by the active stereoisomers of SCH-23390 and butaclamol. Preincubation of NS20Y cells with dopamine induced homologous desensitization of D1 receptor-coupled adenylyl cyclase activity, decreasing dopamine- but not prostaglandin-, adenosine-, or forskolin-stimulated cAMP production. Desensitization did not affect the EC50 for dopamine but resulted in an 85-90% reduction in the maximal response. Dopamine-induced desensitization of adenylyl cyclase activity was found to be both dose and time dependent. As early as 5 min after preincubation with dopamine, cAMP production was decreased by 45-50%, with maximal desensitization occurring by 90 min. Preincubation of NS20Y cells with dopamine also induced a decrease in D1 receptor ligand binding activity, as assessed with the radiolabeled antagonist [3H]SCH-23390. This decrease in binding activity occurred more slowly than the loss of enzyme activity, not achieving maximal levels until after 3 hr. [3H]SCH-23390 saturation binding isotherms in control and maximally desensitized NS20Y cell membranes revealed no change in affinity (KD); however, a 65-70% decrease in receptor number (Bmax) was observed. Because the maximal and temporal decrease in D1 receptors does not correlated with the decrease in dopamine-stimulated enzyme activity, the desensitization may involve a functional uncoupling of the D1 receptor in addition to receptor down-regulation. This is further suggested by a loss in high affinity agonist binding observed in agonist/[3H]SCH-23390 competition experiments after desensitization. Removal of dopamine after maximal desensitization/down-regulation results in recovery to control values by 24 hr. This recovery is mostly, but not completely, blocked by protein synthesis inhibitors, suggesting an involvement of receptor degradation in the desensitization process.

Adenylyl Cyclases↗
PubMed1986

Mechanisms of action of inhibitors of prolactin secretion in GH3 pituitary cells. II. Blockade of voltage-dependent Ca2+ channels.

Pharmacologic agents reported to inhibit prolactin secretion by GH3 pituitary cells were observed to inhibit protein synthesis by these cells in a Ca2+-dependent manner. The possibility that these substances exert their effects on protein synthesis by restricting intracellular Ca2+ availability was explored. Trifluoperazine and chlorpromazine at concentrations that inhibit amino acid incorporation reduced uptake of 45Ca2+ by intact cells approximately 30% under nondepolarizing conditions. Increased extracellular K+ (30 mM), which depolarizes the membrane and opens the voltage-dependent Ca2+ channel of GH3 cells, produced a 2-fold increase in 45Ca2+ uptake; phenothiazines fully suppressed this effect of K+. Nifedipine, verapamil, ergotamine, bromocriptine, (+)- and (-)-butaclamol, and calmidazolium were also effective in inhibiting 45Ca2+ uptake under depolarizing and nondepolarizing conditions. The membrane potential of either depolarized or nondepolarized cells, as determined by [3H]tetraphenylphosphonium+ distribution, was not affected significantly by secretory inhibitors. Increased extracellular K+ altered protein synthesis by GH3 cells in a biphasic manner. Amino acid incorporation by cells incubated at low extracellular Ca2+ concentrations was stimulated by K+, whereas incorporation by cells in high Ca2+ medium was inhibited by K+. Trifluoperazine, chlorpromazine, nifedipine, and bromocriptine suppressed both effects of K+ on protein synthesis. It is proposed that these antagonists of secretion inhibit protein synthesis by GH3 cells through blockade of voltage-dependent Ca2+ channels.

Action Potentials↗
PubMed1989

Characterization of membrane-bound and soluble D2 receptors in canine caudate using [125I]IBZM.

(S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]benzamide (IBZM) was shown to be a high-affinity antagonist selective for the D2 subtype of dopamine receptor. Binding sites for the radioligand [125I]IBZM were characterized with membranes and digitonin-solubilized extracts of canine caudate enriched by chromatography on heparin-agarose. Nonspecific binding, defined using 2 microM (+)-butaclamol, was less than 10% of the total ligand bound at the Kd of the receptor for [125I]IBZM. Direct binding, competition and kinetic experiments indicated that [125I]IBZM bound to a homogeneous population of binding sites. The rank order of potency for inhibition of the binding of [125I]IBZM by antagonists and agonists was found to be consistent with the pharmacological profile expected of a D2 receptor. The affinities of [125I]IBZM for membrane-associated and detergent-solubilized binding sites were essentially identical (Kd congruent to 0.4 nM). This result contrasts with findings obtained in studies with [3H]spiroperidol, where a marked decrease in the affinity of the receptor for the ligand has been observed during detergent solubilization and purification of the receptor. The high selectivity, nanomolar affinity and high specific activity of [125I]IBZM and the results obtained in studies with detergent extracts suggest that [125I]IBZM will be a particularly useful ligand for studies of D2 receptors in the presence of detergents.

Animals↗