Proportion Of Receptors Research Articles

Quantitative systems pharmacology modeling sheds light into the dose response relationship of a trispecific T cell engager in multiple myeloma

In relapsed and refractory multiple myeloma (RRMM), there are few treatment options once patients progress from the established standard of care. Several bispecific T-cell engagers (TCE) are in clinical development for multiple myeloma (MM), designed to promote T-cell activation and tumor killing by binding a T-cell receptor and a myeloma target. In this study we employ both computational and experimental tools to investigate how a novel trispecific TCE improves activation, proliferation, and cytolytic activity of T-cells against MM cells. In addition to binding CD3 on T-cells and CD38 on tumor cells, the trispecific binds CD28, which serves as both co-stimulation for T-cell activation and an additional tumor target. We have established a robust rule-based quantitative systems pharmacology (QSP) model trained against T-cell activation, cytotoxicity, and cytokine data, and used it to gain insight into the complex dose response of this drug. We predict that CD3-CD28-CD38 killing capacity increases rapidly in low dose levels, and with higher doses, killing plateaus rather than following the bell-shaped curve typical of bispecific TCEs. We further predict that dose–response curves are driven by the ability of tumor cells to form synapses with activated T-cells. When competition between cells limits tumor engagement with active T-cells, response to therapy may be diminished. We finally suggest a metric related to drug efficacy in our analysis—“effective” receptor occupancy, or the proportion of receptors engaged in synapses. Overall, this study predicts that the CD28 arm on the trispecific antibody improves efficacy, and identifies metrics to inform potency of novel TCEs.

Is 18F-FDG PET/CT Beneficial for Newly Diagnosed Breast Cancer Patients With Low Proportion of ER Expression?

ObjectiveIt is unclear whether the receptor status of breast malignancy or the proportion of receptors expression is useful in the interpretation of 18F-FDG PET/CT. This study’s purpose was to analyze whether 18F-FDG PET/CT was valuable for helping newly diagnosed breast cancer patients find suspected or unsuspected metastasis lesions based on the proportion of receptors expression.Materials and MethodsEighty newly diagnosed breast cancer patients were divided into six groups, containing N0 (no extraaxillary lymph node metastasis), N1 (extraaxillary lymph node metastasis), M0 (no distant metastasis), and M1 (distant metastasis) groups, C0 (no unsuspected metastasis), and C1 (unsuspected metastasis and treatment plan changed) detected by PET/CT. The main data, including the proportion of receptors ER (estrogen receptor), PR (progesterone receptor), and Her-2 (human epidermal growth factor receptor 2) status, were extracted. Simple correlation and logistic regression were preformed to analyze the association between them.ResultsPatients in N1 group had lower proportion of ER (%) and PR (%) than that in N0 group (ER: 2 [0–80] vs. 80 [15–95]; PR: 1 [0–10] vs. 20 [0–45], p<0.001). Moreover, the proportions of ER and PR were negatively correlated with N1 (ER: [r= −0.339, p= 0.002], PR: [r= −0.247, p= 0.011]) by simple correlation. Also, patients in C1 group had lower proportion of ER (%) and PR (%) than those in C0 group (ER: 10 [0–85] vs. 80 [15–90], p=0.026; PR: 1 [0–10] vs. 20 [0–70], p=0.041), while the distribution of ER and PR between M1 and M0 group had no significant difference. After the adjustment of traditional factors, the negative correlation between the proportion of ER (OR=0.986, 95% CI of OR [0.972–0.999], p=0.016) and C1 was found by logistic regression, cutoff value was 25% (ER) calculated by ROC (Receiver Operating Characteristic) curve (AUC [Area Under Curve]= 0.647, p=0.024).ConclusionThe proportion of ER in newly diagnosed breast cancer was negatively correlated with unsuspected metastasis detected by 18F-FDG PET/CT. 18F-FDG PET/CT might be recommended for newly diagnosed breast cancer patients with single lesions when the ER expression proportion is less than 25% to find unsuspected metastasis lesions and to modify treatment plan contrasted with conventional imaging and clinical examination.

Evaluation of bifurcation phenomena in a modified Shen–Larter model for intracellular $$\hbox {Ca}^{\varvec{2+}}$$ Ca 2 + bursting oscillations

The present work describes an evaluation of the bifurcation phenomena in a modified Shen–Larter model based on calcium-induced calcium release and inositol triphosphate $$(\hbox {IP}_{3})$$ crosscoupling for calcium ion $$(\hbox {Ca}^{2+})$$ bursting oscillations. A time delay for negative $$\hbox {Ca}^{2+}$$ feedback on the $$(\hbox {IP}_{3})$$ receptor is added to the original Shen–Larter model, by introducing the proportion of receptors not inactivated by $$\hbox {Ca}^{2+}$$ as a new variable. Compared with the original model, the number of chaotic regions for a stimulation level r is significantly reduced, and regions of $$\hbox {Ca}^{2+}$$ oscillations (particularly bursting) appear to become slightly enlarged. Different topological types of bursting oscillations in this modified model are classified by fast/slow dynamical analysis and codimension-2 bifurcations of fast subsystem, when choosing better of two slow variables the free $$\hbox {Ca}^{2+}$$ concentration in the endoplasmic reticulum and the $$\hbox {IP}_{3}$$ concentration in the cytosol. Furthermore, classification and transition mechanisms of bursting $$\hbox {Ca}^{2+}$$ oscillations could help to understand or detect more distinctive oscillatory behaviors of real cells in response to different levels of stimulation.

Ex vivo [11C]‐(+)‐PHNO binding is unchanged in animal models displaying increased high‐affinity states of the D2receptor in vitro

Dopamine (DA) D2 receptor supersensitivity has been linked to an increase in the density of the D2 high-affinity state as measured in vitro. The two- affinity-state model of the D2 receptor predicts that the ex vivo specific binding of [11C]-(+)-PHNO, an agonist radiotracer thought to bind selectively to the high-affinity state in vivo, should be increased in animal models that display in vitro increases in the proportion of receptors in the D2 high-affinity state. Here, we test this hypotheses by comparing the ex vivo SBR of [11C]-(+)-PHNO with that of the antagonist radiotracer [3H]-raclopride in three dopaminergically supersensitive rat models-AMPH-sensitized rats, rats withdrawn from chronic ethanol, and unilaterally 6-OHDA-lesioned rats-using ex vivo dual-radiotracer biodistribution studies. We find that in AMPH-sensitized rats and rats withdrawn from chronic ethanol treatment, models that exhibited approximately 4-fold increases in the D2 high-affinity state in vitro, the SBRs of [11C]-(+)-PHNO and [3H]-raclopride are unchanged relative to control rats. In unilaterally 6-OHDA-lesioned rats, we find that the increase in [11C]-(+)-PHNO SBR is no different than that observed for the antagonist radiotracer [3H]-raclopride (54% +/- 16% and 52% +/- 14%, respectively). In addition, the effect of acute AMPH pretreatment (4 mg/kg, i.v.) on the SBRs of [11C]-(+)-PHNO and [3H]-raclopride is equivalent in AMPH-sensitized (-38% +/- 12% and -36% +/- 8%, respectively) and in control rats (-40% +/- 11% and -38% +/- 7%). These data emphasize a significant discrepancy between in vitro and in vivo measures of D2 agonist binding, indicating that the two-affinity-state model of the D2 receptor may not apply veridically to living systems. The potential implications of this discrepancy are discussed.

GABA receptor proteins within lipid rafts in the AY‐9944 model of atypical absence seizures

The inhibition of cholesterol synthesis with AY-9944 (AY) results in chronic recurrent atypical absence seizures in rodents. We hypothesized that cholesterol inhibition during the course of creating the AY model of atypical absence seizures results in an alteration of the entry of gamma-aminobutyric acid (GABA)(A) and GABA(B) receptors into lipid rafts that contributes to epileptogenesis in this model. The cholesterol synthesis inhibitor AY (7.5 mg/kg) was administered on postnatal day (P) 2, P8, P14, and P20 in Long-Evans hooded rats. The incorporation of GABA(A) and GABA(B) receptor proteins into lipid rafts of the brain was then determined. AY produced a shift of both GABA(A) and GABA(B) receptors in the examined detergent-resistant membranes (DRMs) and the soluble fractions. The percentage of the GABA(A) and GABA(B) receptors that shifted out of the DRMs varied between 17% and 50%, but the proportion of receptors in DRMs were decreased to levels around that of P5 animals or even lower. The shift observed in the AY-treated versus control animals was statistically significant (p < 0.01) for both GABA(A) and GABA(B) receptors. Cholesterol synthesis inhibition during rat brain development that is induced by AY leads to chronic atypical absence seizures and is associated with an alteration of GABA(A) and GABA(B) receptor proteins within lipid rafts. These data suggest a novel avenue of investigation into the epileptogenesis of experimental chronic atypical absence seizures.

Effects of transient reduction of endogenous 5-HT levels on 11C-DASB and 11C-MDL100907 specific binding: Implications for finding a serotonergic radiotracer vulnerable to endogenous competition in humans

Serotonin (5-HT) neurotransmission is involved in the normal regulation of affect and cognition, and its dysfunction is implicated in a range of mental disorders. However, there is currently no method to directly assess 5-HT synaptic levels in the living human brain. In the human dopamine (DA) system, baseline receptor occupancy by endogenous DA can be estimated by measuring the effects of DA depletion on the binding of certain radioligands through endogenous competition, particularly in striatum. For example, the measured increase in 11C-raclopride and 123I-IBZM binding following acute DA depletion implies a baseline D2 receptor occupancy of 10% in healthy humans. Brain 5-HT can be transiently and safely reduced by rapid tryptophan depletion (RTD), a technique involving the ingestion of an amino acid mixture lacking L-tryptophan, the dietary precursor of 5-HT. We have used RTD in healthy humans in an attempt to demonstrate a measurable effect of reduced 5-HT on the specific binding of the selective 5-HT transporter (SERT) ligand 11C-DASB and the selective 5-HT2A receptor antagonist ligand 11C-MDL100907. However, values of regional specific binding were not significantly different between the reduced endogenous 5-HT and control conditions for either tracer. These results suggest that PET with 11C-DASB or 11C-MDL100907 is not sensitive to the decreased concentration of synaptic 5-HT following RTD, and that neither tracer has the potential for measuring changes in synaptic levels of 5-HT in vivo. Imaging techniques relying on endogenous competition would ideally require a brain region with high synaptic levels of, and significant baseline receptor occupancy by, endogenous neurotransmitter. However, in vivo microdialysis studies in non-human primates suggest that extracellular 5-HT concentration is very low (< 0.5 nM) across brain regions and is 1 – 2 orders of magnitude lower than striatal DA levels. In the case of the 5-HT2A receptor, this concentration is significantly below the estimated KD of 5-HT for the receptor configured in a state of high affinity for agonists ( 1.3 nM). Moreover, in vitro studies suggest that the proportion of receptors configured in a state of high affinity for agonists, and therefore potentially available to endogenous 5-HT, is low ( 13%). Together, these values suggest that baseline occupancy of 5-HT2A receptors by 5-HT may be only approximately 2%, making changes following increased or decreased 5-HT unmeasurable by current techniques in humans. Agonist serotonergic radioligands may be expected to be superior for endogenous competition studies, but have so far been unsuccessful in humans. Efforts to experimentally increase baseline receptor occupancy (for example by pretreatment with selective 5-HT reuptake inhibitor medication) prior to RTD may be of interest. However, the insensitivity of current tracers to endogenous competition implies that they may be used to measure differences in SERT or 5-HT2A receptor availability between populations or during therapy without the need to consider concomitant changes in neurotransmitter concentration.

Nicotine is highly effective at producing desensitization of rat alpha4beta2 neuronal nicotinic receptors.

We examined desensitization by acetylcholine (ACh) and nicotine at the rat alpha4beta2 neuronal nicotinic receptor stably expressed in HEK cells. For both agonists, the decay in response due to desensitization ('onset') was best fitted by the sum of two exponentials with the fast component dominant at concentrations > 1 microM. The time constants for onset were similar for both agonists, and showed little concentration dependence over the range of 0.1-100 microM. Recovery from desensitization also showed two exponential components. In contrast to the similarity in onset, nicotine produced longer lasting desensitization, resulting from an increase in the proportion of receptors in the slowly recovering population and from an increase in the time constant for the slow recovery process. The proportion of receptors in the slowly recovering population increased as the duration of the desensitizing pulse increased. Desensitization was also induced by low concentrations of agonist, with no apparent macroscopic response. A 100 s application of 10 nM nicotine desensitized 70 % of the peak response, while 100 s of 10 nM ACh desensitized only 15 %. At higher concentrations of agonist, which result in a macroscopic response, desensitization in the absence of activation also can occur. Nicotine is a very potent and efficacious desensitizing agent at this neuronal nicotinic receptor.

Acetylcholinesterase promotes neurite elongation, synapse formation, and surface expression of AMPA receptors in hippocampal neurones

Here we show that chronic application of low concentrations (0.01–0.05 U/ml) or a single application of 1–5 U/ml acetylcholinesterase (AChE) promotes the extension of neuronal processes, synapse formation, and α-amino-3-hydroxy-5-methylisoxazolepropionate receptor (AMPAR) surface expression in both embryonic and postnatal hippocampal cultures. The total number of AMPARs was unchanged but the proportion of receptors that were surface-expressed, predominantly at synapses, was approximately doubled following AChE treatment. Blockade of the peripheral anionic site of endogenous AChE in the cultures dramatically reduced neurite outgrowth but did not alter the appearance of synaptic markers SV2a and PSD95. These results indicate that AChE is necessary for normal dendrite and axon formation in hippocampal neurones and suggest that it may also play a role in excitatory synapse development, plasticity, and remodelling.

Quantitative immunolocalization of mu opioid receptors: regulation by naltrexone

The present study utilized a newly developed quantitative immunohistochemical assay to measure changes in mu opioid receptor abundance following chronic administration of the opioid receptor antagonist naltrexone. These data were compared with those obtained from mu receptor radioligand binding on adjacent tissue sections, in order to determine whether the characteristic antagonist-induced increase in radioligand binding is due to an increase in the total number of mu receptors and/or to an increase in the proportion of receptors that are in an active binding conformation in the absence of a change in the total number of receptors. Adult male Sprague–Dawley rats were administered naltrexone, 7–8 mg/kg per day, or saline continuously for seven days by osmotic minipumps, after which time their brains were processed for immunohistochemistry and receptor autoradiography on adjacent fresh frozen tissue sections. Semiquantitative immunohistochemistry was performed using a radiolabelled secondary antibody for autoradiographic determination and a set of radioactive standards. Results demonstrate an overall concordance between the distribution of mu opioid receptors as measured by the two different methods with a few exceptions. Following naltrexone administration, mu receptor immunoreactivity was significantly higher in the amygdala, thalamus, hippocampus, and interpeduncular nucleus as compared with the saline-treated control animals. [ 3H]D-Ala 2,N-Me-Phe 4,Gly-ol 5-enkephalin binding to mu opioid receptors was significantly higher in the globus pallidus, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, ventral tegmental area, central gray, and interpeduncular nucleus of the naltrexone-treated rats. These findings indicate that in some brain regions chronic naltrexone exposure increases the total number of mu opioid receptors, while in other regions there is an increase in the percent of active receptors without an observable change in the total number of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of mu opioid receptors on tissue sections.

Relation of chemical structure to spatial distribution of sensory responses in rat olfactory epithelium.

1. Electroolfactogram (EOG) recordings were made in three configurations from the rat olfactory epithelium. Each configuration compared recordings in the dorsomedial recess of the epithelium with recordings in ventral or lateral parts of the epithelium. Most comparisons were made with simultaneous recordings. The exception was a series in which the dorsal recess and lateral space between the base of two turbinate bones were directly exposed for odor application and recording. The spatial distributions of maximal responses were largely independent of recording configuration. 2. Simultaneous recordings compared dorsomedial and lateral sites in the epithelium during stimulation with a series of 50 odorants. The odorants that evoked larger responses in the lateral sites were usually compounds that lacked oxygen containing functional groups (such as the carbonyl group). This was true for straight chain and cyclic alkanes, for terpine compounds, and for aromatic compounds. The major exception was cineole, a bicyclic compound. All compounds containing ketone groups evoked larger dorsomedial responses. The responses of aldehydes and esters depended upon whether they were attached to aliphatic or aromatic chains. 3. In the three types of preparation, the sites responding best to ketones were in the same expression zone of the epithelium according to published maps for the rat and mouse. The sites responding best to odors without functional groups were in the far lateral or ventral region and corresponded to one of the two most lateral and ventral expression zones. This fact suggests that the receptors in these regions have a preference for particular chemical properties. This level of analysis cannot determine whether all receptors in each zone have a stronger response to certain properties of these odorants or whether each zone contains different proportions of receptors with these properties.

D2 receptor antagonists do not modify guanine nucleotide-sensitive interactions between dopamine and D1 dopamine receptors under in vitro conditions.

This study investigated possible D1/D2 interactions in rat and bovine striatal tissue by examining the effects of D2 antagonists on the action of dopamine at D1 dopamine receptors. In addition, the extent to which D2 antagonists may induce an agonist low-affinity state of the D1 receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D1 receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp (NH)p, was not altered by pretreatment with either of the selective D2 antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D1 receptors, and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 microM) but not by eticlopride. A molar excess of dopamine (100 microM) promotes the dissociation of (+/-)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-o l ([3H]SCH 23390) from rat striatal D1 receptors at a rate that is significantly slower than when dissociation is initiated using 1 microM piflutixol. After pretreament with Gpp(NH)p, [3H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [3H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D1 receptors that are Gpp(NH)p sensitive and D2 antagonist insensitive and provide no evidence to support a D1/D2 link operating at the receptor level.

In-vivo binding of (+)-[3H]PN 200-110 to peripheral tissues and brain of spontaneously hypertensive rats: effect of lacidipine.

The time-course of dihydropyridine receptor occupancy by lacidipine and its relationship with pharmacological activity has been studied in spontaneously hypertensive rat (SHR), as measured by the inhibition of specific (+)-[3H]PN 200-110 binding in-vivo. After oral administration of doses active in reducing blood pressure, lacidipine did not show tissue target differences in respect to binding sites labelled by (+)-[3H]PN 200-110 in cerebral cortex, heart, ileum, bladder and thoracic aorta. The relative occupancy of receptors in heart 60 min after oral administration of 1 mg kg-1 lacidipine was 75%. After 12 h, when lacidipine was still effective in reducing blood pressure in SHR, a low (15%) but detectable proportion of receptors was still occupied by the drug. The percentage decrease of blood pressure was linear with the percentage of receptor occupancy obtained by different doses of lacidipine; that is, there was a close correspondence between ED25 for decrease in blood pressure (0.33 mg kg-1) and ED25 for inhibition of (+)-[3H]PN 200-110 specific binding in the heart (0.36 mg kg-1). The long-lasting effect of lacidipine on blood pressure might be explained by its selective interaction with dihydropyridine binding sites labelled in-vivo by (+)-[3H]PN 200-110.

Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin

1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.

A 1 adenosine receptors in the human fat cell: tissue distribution and regulation of radioligand binding

Identification of adenosine A 1 receptor binding sites was performed using the antagonist. [ 3H]1,3-dipropyl-8-cyclopentyl-xanthine ([ 3H]DPCPX), and the agonist [ 3H](−)-N 6-R-phenylisopropyladenosine ([ 3H](−)-PIA) on human adipocyte membranes from different anatomical localizations (i.e. abdominal, femoral and omental fat deposits). Despite the strong antilipolytic effect initiated by various adenosine analogs, the human adipocytes were poorly equipped in adenosine A 1 receptors whatever the anatomical location (B max ⩽ 95 fmol/mg of protein for the antagonist [ 3H]DPCPX and ⩽ 72 fmol/mg for the agonist [ 3H](−)-PIA). There was no marked difference between the three fat deposits in terms of maximal binding for both radioligands. Saturation and competition experiments showed that the proportion of receptors in the high-affinity state for the agonists was very high (70–91%), but only 33–44% of them were guanine nucleotide-sensitive. Moreover the guanine nucleotides were shown to enhance the specific binding of the antagonist [ 3H]DPCPX by decreasing its K D value. These binding properties are strongly different from those of another G i-coupled receptor on the human fat cell, the α 2A-adrenoceptor, and indicates that the adenosine A 1 receptor and the α 2-adrenoceptor could be differentially coupled with G 1 proteins in the human fat cell.

Differential sensitivity of the insulin receptor to proteolysis after β-adrenergic stimulation

The cellular mechanism by which the specific binding of [ 125I]insulin to intact rat adipocytes is inhibited by isoproterenol has been studied. By exposing control and isoproterenol-treated cells to trypsin (0–150 μg/ml for 20 min at 4°C) and measuring the intact insulin receptor pool following detergent solubilization, a differential sensitivity to proteolysis of the cell membrane receptor was observed. At low trypsin concentration (< 30 μg/ml), approximately 40% of the specific insulin binding in isoproterenol-treated cells was insensitive to proteolysis as compared to control cells. At higher levels of trypsin (50–150 μg/ml) both groups displayed similar levels of trypsin-insensitive receptors which, at the highest trypsin concentration, accounted for 10% of the total receptors in intact cells. Detergent-solubilized receptors from isoproterenol-treated cells, on the other hand, exhibited the same sensitivity to trypsin proteolysis as solubilized receptors from control cells. The time course of the onset and reversal of the isoproterenol-induced binding alteration in intact adipocytes has been analyzed by mild trypsinization (20 μg/ml). Results indicated that insulin receptors resistant to trypsin under these conditions mediated the decreased surface binding and were re-expressed on the cell surface upon removal of isoproterenol. Experiments in which adipocytes were fractionated into plasma membrane and Golgi-enriched fractions indicated that the loss of surface insulin binding was not accompanied by a decrease in the proportion of receptors in the adipocyte plasma membrane. These results suggest that the isoproterenol-induced decrease in surface insulin receptor sensitivity to mild trypsinization may be a result of a modification or sequestration of receptors in a membranous compartment in proximity or continuity with the adipocyte plasma membrane.

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms.

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.

High and low affinity states of the dihydropyridine and phenylakylamine receptors on the cardiac calcium channel and their interconversion by divalent cations

Drug receptors associated with Ca 2+-channels in isolated chick heart membranes were found to exist in high and low affinity states. When assays were conducted in the presence of EDTA most of the receptors detected with the dihydropyridines (+)[ 3H]PN 200-110 or [ 3H]nitrendipine appeared to be in the lower affinity state. Inclusion of either Mg 2+ or Ca 2+ in the binding reactions resulted in the disappearance of the lower affinity state and the conversion of the receptors to a single high affinity state. Similar results were obtained with the phenylalkylamine derivative [ 3H]desmethoxyverapamil (D888). The results suggest that both the dihydropyridine and phenylalkylamine receptors on the cardiac Ca 2+-channel can exist in interconvertible high and low affinity states in vitro , and that the proportion of receptors in each affinity state can be altered by the absence or presence of divalent cations.

Uncoupling between beta-adrenoceptors and adenylate cyclase in dog ischemic myocardium.

We evaluated the effects of ischemic injury on the myocardial adenylate cyclase system, 5 h after ligation of the left anterior descending coronary in 5 anesthetized dogs. Crude cardiac membrane preparations were isolated from control and ischemic areas of ventricular myocardium and tested for: 1. L-(125I)iodocyanopindolol binding, in the absence and presence of +/- -isoprenaline and GTP, and 2. adenylate cyclase activity. The density of beta-adrenoceptors increased by 35% in membranes from ischemic areas while the proportion of receptors in a high affinity state for +/- -isoprenaline decreased from 43% to 20%. Adenylate cyclase activities in the basal state and under stimulation with NaF, forskolin, Gpp(NH)p, +/- -isoprenaline and VIP were all markedly and similarly reduced, being only about 30% of comparable activities in membranes from control areas. The +/- -isoprenaline subsensitivity of cardiac adenylate cyclase can, thus, be attributed to a defective enzymatic system and not to a reduction in the number of beta-adrenoceptors implying that the internal components of the system were more sensitive to acute ischemia than the outward oriented hormone receptors. It is tempting to ascribe this uncoupling to a functional depletion in the guanine nucleotide-binding regulatory protein Ns that might reflect a loss of high energy phosphate stores including GTP.

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.

The binding of rat uterine cytosol oestrogen receptors to oligodeoxythymidylate--cellulose. Its relationship to a stable form of receptor complex with separate ligand- and oligonucleotide-binding sites.

The interaction of rat uterine cytosol oestrogen-receptor complexes with the synthetic acceptor oligo(dT)--cellulose was studied. Differences in the stability of receptor complexes and their ability to bind to oligo(dT)--cellulose on storage at 4 degrees C or when exposed to increased temperatures indicated heterogeneity of steroid- and oligonucleotide-binding sites. Dilution, dialysis and (NH4)2SO4 precipitation increased the interaction of receptor complexes with oligo(dT)--cellulose (a step termed activation). This increase may be the result of the removal of low-molecular-weight cytosol components which inhibit receptor activation, dimerization to the 5 S form, which binds to oligo(dT)--cellulose, or interaction of 5 S receptor with the oligonucleotide. Cytosol oestradiol--receptor complexes exhibited biphasic dissociation kinetics. All these manipulations resulted in an increase in the proportion of the slow-dissociating component equivalent to the increase in receptor binding to oligo(dT)--cellulose. In contrast, addition of 10mM-sodium molybdate to cytosol decreased both oligo(dT)--cellulose binding and the proportion of receptor with slow dissociation kinetics. The inclusion of proteinase inhibitors did not affect interactions of receptor with oligo(dT)--cellulose nor the dissociation kinetics. These results suggest that oligo(dT)--cellulose binding may serve to quantify the proportion of cytosol receptor in an active form capable of nuclear interaction and to help to ascertain whether a receptor system is fully functional. This binding procedure could prove useful in the evaluation of oestrogen responsivity under normal and pathological conditions.