Membrane Receptor Density Research Articles

Experimental in vitro phenotype reprogramming of two subsets of neutrophilic granulocytes in children with acute destructive pneumonia by means of a synthetic hexapeptide

Effector dysfunctions of neutrophil granulocytes are often associated with the occurrence of dysregulatory processes in the antibacterial immune defense. Acute destructive pneumonia is a severe purulent-inflammatory disease associated with discordant functions of effector mechanisms of neutrophil granulocytes and emergence of negatively transformed cell subsets. Therefore, the search for new experimental approaches aimed at re-orientation of negatively altered phenotype of distinct subsets of neutrophilic granulocytes in the children with acute destructive pneumonia by means of various immunotropic substances is quite relevant. The aim of the study was to evaluate the modulating effects of synthetic hexapeptide (Arginyl-alpha-Aspartyl-Lysyl-Valyl-Tyrosyl-Arginine) on the contents and phenotype of 2 functionally significant subsets of major (CD16+CD64-CD32+CD11b+) and minor (CD16+CD64+CD32+CD11b+) subpopulations of neutrophils in a closed in vitro experimental system sampled in the children with atypical acute destructive pneumonia. We have examined twenty peripheral blood samples from 10 children with acute destructive pneumonia, and 40 blood samples of 20 healthy children 2-4 years old. Immunophenotyping of neutrophil granulocytes classified in 2 subsets was performed on the basis of expression density of membrane receptors, according to MFI criteria. Phenotypic features of neutrophil granulocyte subsets were evaluated in the in vitro system before and after incubation of peripheral blood with Hexapeptide (10-6 g/L; 37 C, 60 min). In children with acute destructive pneumonia, compared with conditionally healthy children, the following variants of negative transformation of the neutrophil subsets were established: a significant decrease in the ratios of the major subset, i.e., from 98.0 (96.9-98.7) % o 55.8 (35.3-74.8) %, with a decreased CD16 and CD11b density expression according to MFI, and a significantly increase ratio of the minor neutrophil subset: from 1.3 (0.4-1.6) % to 52.6 (41.8-54.9) %, with increased expression of CD11b receptor, and a decrease in CD64 expression. Immunomodulatory effects of Hexapeptide upon neutrophil granulocytes of children with acute destructive pneumonia have been demonstrated in the closed in vitro system showing positive phenotype remodeling of both cell subsets in the absence of significant quantitative changes. Thus, upon treatment with the hexapeptide, we have found a significantly increased expression of activation receptors CD16, CD11b in the major subset, and a significant decrease in their expression for the minor subset to the levels typical to healthy children. At the same time, hexapeptide did not affect the studied subsets of neutrophils from healthy children, except of increased CD64 expression in the minor subset. The obtained data can be used in future to develop new approaches to the targeted immunotherapy aimed at correcting the phenotype of neutrophil granulocyte subsets in acute destructive pneumonia in children.

Impaired αGDI Function in the X-Linked Intellectual Disability: The Impact on Astroglia Vesicle Dynamics.

X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice astrocytes, devoid of pathologic tissue environment, exhibit vesicle mobility deficits. Contrary to previous studies, we show that astrocytes express two GDI proteins. The siRNA-mediated suppression of expression of αGDI especially affected vesicle dynamics. A similar defect was recorded in astrocytes from the Gdi1 -/Y mouse model of XLID and in astrocytes with recombinant mutated human XLID αGDI. Endolysosomal vesicles studied here are involved in the release of gliosignaling molecules as well as in regulating membrane receptor density; thus, the observed changes in astrocytic vesicle mobility may, over the long time-course, profoundly affect signaling capacity of these cells, which optimize neural activity.

Single Molecule Imaging of M2 Muscarinic Receptors in Live Heart Explants

TIRF microscopy allows us to visualize individual fluorophores in living cells. We have developed automated algorithms to detect and track thousands of individual molecules and have studied GPCRs (G-protein coupled receptors) tagged with fluorescent ligands or fluorescent proteins in live cultured cells and freshly dissected tissue slices. We have found that M1 and M2 muscarinic receptors diffuse freely at the plasma membrane with their mobility dependent upon both cell-type and temperature. The mobility of M2 receptors tagged with the fluorescent antagonist (Cy3B-telenzepine) matches the mobility of M2-GFP molecules imaged under the same conditions. Fluorophore tracking allowed direct observation of transient homo-dimerisation of M1 and M2 receptors which we confirmed using two-color imaging. At a membrane receptor density of 2 µm−2, 30% of receptors were dimeric and the dimer lifetime was ∼1 s−1. We have found that only ∼10% of isolated primary cardiomyocytes and cells in the mice heart slices expressed M2 receptors on the detectable level. The receptors undergo unrestricted diffusion and therefore were evenly distributed across the cell membrane. The M2 density in freshly isolated embryonic cardiomyocytes was ∼1 μm−2, increasing at birth to ∼3 μm−2 and decreasing back to ∼1 μm−2 after birth. Whilst some M2 receptors formed reversible dimers the majority were monomeric. The receptor mobility was approximately 4-times faster in freshly dissected heart slices (0.6 µm2 s−1) than in cultured primary cardiomyocytes. Knowing receptor mobility and density we used Monte Carlo simulations to estimate an encounter rate of 5-10 collisions per second. This may explain the observed electrophysiological latency between the application of acetylcholine and GIRK channel opening.

Four Missense Mutations in the Ghrelin Receptor Result in Distinct Pharmacological Abnormalities

The growth hormone secretagogue receptor (GHSR) plays an important role in regulating food intake and energy homeostasis. In this study, we compared the pharmacological properties of four reported variants of the human GHSR (I134T, V160M, A204E, and F279L) with those of the wild-type receptor. Corresponding recombinant receptors were transiently expressed in either human embryonic kidney 293 or COS-7 cells. Basal as well as ligand-induced signaling was assessed by luciferase reporter gene assays and measurement of inositol phosphate production. In addition, receptor expression levels were monitored by whole-cell enzyme-linked immunosorbent assay. Ligand-independent signaling of the wild-type GHSR is significantly reduced with introduction of either the V160M or F279L substitutions, whereas basal activity of the A204E mutant is not detectable. Ghrelin potency is markedly increased at the V160M mutant, whereas the I134T variant is unresponsive to this endogenous agonist. In contrast, the I134T mutant responds to a known GHSR inverse agonist, [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P (SP-analog), albeit with reduced efficacy. Activity of the SP-analog at the V160M and F279L mutants is comparable to the wild type (WT) value. The overall expression level of each of the four GHSR variants is reduced relative to WT; however, the ratio between the intracellular and plasma membrane receptor density remains comparable. Treatment with the SP-analog significantly increases cell surface expression of each receptor with the exception of the A204E variant. Taken together, our studies reveal that naturally occurring GHSR mutations affect a wide range of pharmacologic properties. The physiological impact of these alterations within selected populations (e.g., obese, lean individuals) as well as the pharmacogenomic consequences of corresponding mutations remain to be further investigated.

Chronic hypoxia up-regulates expression of adenosine A 1 receptors in DDT 1-MF2 cells

As the first step to understand how chronic hypoxia might regulate smooth muscle function in health and disease, we have employed an established immortalised cell model of smooth muscle, DDT 1-MF2 cells, to address the hypothesis that adenosine A 1 receptor density is modulated by O 2 availability. Maximal specific binding ( B max) of the selective adenosine A 1 receptor antagonist, [ 3 H ]-DPCPX, to cell membranes increased 3.5-fold from 0.48±0.02 pmol/mg to 1.7±0.5 pmol/mg protein after 16 hr of hypoxia and this effect was not accompanied by any statistically significant changes in either binding affinity (0.84±0.2 nM vs. 1.2±0.3 nM) or Hill coefficient (1.1±0.1 vs. 0.99±0.03). Hypoxia-evoked increases in membrane receptor density were paralleled in intact DDT 1-MF2 cells. In addition, the increase in [ 3 H ]-DPCPX binding to intact cells was inhibited by co-incubation during hypoxia with the translational inhibitor cycloheximide, the transcriptional blocker actinomycin D and the NFκB inhibitor sulphasalazine. Together, these data show that adenosine A 1 receptor density is modulated, at least in part, by O 2-dependent activation of the transcription factor NFκB and adds to the list of processes dynamically regulated by ambient oxygen availability. Since hypoxia is an initiating factor in acute renal failure, similar changes in transcription may account for up-regulation of adenosine A 1 receptors noted previously in the renal vasculature of rats with acute renal failure.

Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese hamster ovary cells expressing human nociceptin/orphanin FQ receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the Gi-coupled N/OFQ receptor (NOP). We have examined the effects of chronic exposure of Chinese hamster ovary cells expressing the recombinant human NOP receptor (CHOhNOP) to 1 nM N/OFQ for up to 48 h in the absence and presence of the NOP selective antagonist [Nphe1]N/OFQ (1–13)NH2 ([Nphe1]). Then, either a concentration–response curve for N/OFQ inhibition of cAMP formation was constructed or the cells were homogenized and membrane receptor density was determined using [125I]Y14N/OFQ. There was a time-dependent reduction in pEC50 (without a change in maximum) for N/OFQ with significant differences observed following >24 h of exposure (control pEC50∼9.5; 48 h pretreatment∼8.7). In cells co-exposed to N/OFQ+[Nphe1] for 48 h, there was no reduction in pEC50. There was a compensatory (∼2.5-fold), [Nphe1]-sensitive increase in cAMP mass in cells exposed to N/OFQ for 24–48 h. N/OFQ pretreatment also resulted in a time-dependent [Nphe1]-sensitive loss of cell surface receptors. At 48 h, Bmax was reduced from ∼2.0 to ∼1.3 pmol mg−1 protein without a change in pKd for N/OFQ. There was a positive correlation between pEC50 for cAMP inhibition and Bmax. The lack of effect on maximum cAMP response probably results from receptor overexpression and the creation of a receptor reserve.

Regulation of parathyroid hormone/parathyroid hormone-related protein receptor expression by osteoblast-deposited extracellular matrix in a human osteoblast-like cell line.

Parathyroid hormone (PTH) receptors and the biological response to PTH in osteoblasts have been shown to be influenced by glucocorticoids, growth factors, cytokines or PTH itself. Furthermore, components of extracellular matrix (ECM) appear to regulate the response to PTH as well. We investigated the effects of osteoblast-deposited ECM on PTH-related protein (PTHrP)-stimulated cAMP production, PTHrP binding and PTH/PTHrP receptor mRNA in the human osteoblast-like cell line SaOS-2. ECM was laid down by the human osteoblastic cell line MG-63. At confluence, maximal cAMP stimulation induced by 100 nmol/l PTHrP (1-34) was decreased in SaOS-2 cells grown on ECM as compared with cultures on plastic dishes, without any change in PTHrP concentration producing half-maximal stimulation. In contrast, cAMP production stimulated by PGE2 was increased in cells on ECM. Saturable 125I-PTHrP binding (as evaluated by Scatchard plot analysis) was markedly diminished in cells grown on ECM (5,600 +/- 2,010 vs. 20,700 +/- 1,710 binding sites/cell, x +/- S.E.M., P < 0.01, n = 4 experiments), without any significant change in affinity (1.3 +/- 0.4 vs. 2.5 +/- 0.5 nmol/l (NS), in cells on ECM and plastic, respectively). This apparent decrease in membrane receptor density was associated with markedly lower steady state PTH/PTHrP receptor mRNA levels as assessed by Northern blot analysis (ECM/control: 0.4 +/- 0.1). A difference in PTH/PTHrP receptor mRNA levels between cells on ECM or on plastic dishes was detectable by 8 hours but not by 4 hours, after seeding the cells at high density. By 24 hours after plating, PTH/PTHrP receptor mRNA levels were maximally decreased in cells on ECM. These results in the human osteoblast-like cell line SaOS-2 indicate that PTH/PTHrP receptors are down-regulated by growth on ECM. Thus, attachment of bone cells to bone surface could influence differentiation and function of osteoblasts.

Determination of agonist dissociation constants in isolated vasculature: Equivalence of estimates obtained by the method of partial irreversible receptor inactivation and a novel application of the operational model of pharmacological agonism

Steady-state contractile responses elicited by activation of the ∞ 1-adrenergic and 5-HT 2 receptors in isolated rat and rabbit aorta, respectively, were analyzed. Agonist dissociation constants (K A's) obtained by the method of partial irreversible receptor inactivation were compared to K A values determined by fits of the operational model of pharmacological agonism to single concentration response curves (CRCs). The observed nature of the K A estimates obtained with the Furchgott method for phenylephrine (PE) and oxymetazoline (OXY) at the ∞ 1-adrenergic receptor and for 5-hydroxytryptamine (5-HT) at the 5-HT 2 receptor in isolated rat aorta, and for PE and 5-HT at the ∞ 1-adrenergic and 5-HT 2 receptors, respectively, in isolated rabbit aorta, was consistent with the hypothesis that the density of membrane receptors is greatly in excess of the density of transducer proteins (i.e., [R o] ⪢ [T o]) in these systems. Therefore, K A, efficacy and slope factor estimates were also obtained by computer fits of the operational model to single agonist CRCs in both rat and rabbit aorta, with the empirically determined tissue maximal response (T max) substituted for the theoretical parameter E m. In all cases, the mean pK A estimates obtained with the operational model closely approximated and were strongly correlated with the mean pK A estimates determined by the Furchgott method. These studies suggest that, at least in some vascular preparations T max is a good estimate of E m, and moreover, that E m may be not only a specific characteristic of a given receptor-effector system as previously demonstrated by Black and Leff, but that E m may also describe a more general feature of tissue responsiveness that is shared among distinct membrane receptors coupled to similar effector systems. In conclusion, when receptor inactivation studies have indicated that the condition [R o ⪢ [T o] exists, T max can be substituted into the operational model to provide valid estimates of agonist K A values at distinct receptor subtypes, in the absence of receptor alkylation.

Enhancement of adenosine A2 and prostaglandin E1 receptor-mediated cAMP generation by prior exposure of Swiss 3T3 fibroblasts to Ca2+-mobilizing receptor agonists or phorbol ester: Activation of protein kinase C triggers increases in the receptor density in cell membranes

Production of cAMP in response to adenosine A2 or prostaglandin E1 receptor stimulation was, but the production induced by a beta-adrenergic agonist or forskolin was not, enhanced by prior exposure of Swiss 3T3 fibroblasts to agonists of Ca2+-mobilizing receptors or phorbol ester for 3 h. The enhancement reflected potentiation of the receptor-coupled activation of adenylate cyclase and the 2-fold increase in the adenosine A2 receptor number in membranes under these conditions. No enhancement was observed, however, when the medium used for the prior exposure was further supplemented with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporin, inhibitors of protein kinase C, neither of which affected the cAMP responses of the nonexposed cells. It is very likely, therefore, that activation of protein kinase C triggers the increase in certain receptor density in membranes, thereby enhancing the receptor-coupled cAMP-generating responses. The physiological significance of such cross-talk between cellular signaling systems is discussed in comparison with similar previous observations.

Predominance of β-adrenergic over α-adrenergic receptor functions involved in phosphorylase activation in liver cells of cholestatic rats

Hepatocytes isolated from normal and cholestatic rats responded to adrenergic agonists and antagonists in a quite different manner. Much greater activation of glycogen phosphorylase was caused by phenylephrine, an α-agonist, than by isoproterenol, a β-agonist, in normal rat hepatocytes, and vice versa in the cholestatic rat cells. Epinephrine activation of phosphorylase was antagonized more efficiently by phenoxybenzamine, an α-antagonist, than by propranolol, a β-antagonist, in normal rats, whereas it was antagonized totally by propranolol but only partially by phenoxybenzamine in cholestatic rat hepatocytes. The number of α-adrenergic receptors, measured by [ 3H]prazosin binding to membranes, as well as α-receptor-mediated increases in 32 P i incorporation into phosphatidylinositol and in 45Ca efflux, were reduced in hepatocytes after induction of cholestasis. The reduction of these parameters of α-receptor-linked functions was associated with the reciprocal increase in the number of β-receptors and enhancement of β-receptor-mediated accumulation of cyclic AMP in cholestatic rat hepatocytes. The affinity of epinephrine for β-receptors was higher in cholestatic rat cells than in normal rat cells; this difference in affinity was abolished by the addition of guanylylimidodiphosphate, indicating that induction of cholestasis rendered hepatic β-receptors more tightly coupled to the GTP-binding protein. Thus, the cascade reactions arising from β-receptors are predominant over those from α-receptors, eventually leading to glycogen breakdown in cholestatic rat hepatocytes, principally because of not only the elevated β to α ratio of the membrane receptor density but also the tight coupling of β-receptors to the adenylate cyclase system via the guanine nucleotide regulatory protein.

Regulation of acetylcholine receptor density in membranes of denervated mouse muscles.

The ACh sensitivity of denervated muscles of rats stimulated in situ or in culture for several days decreases to low levels characteristic of normal muscles. Possible causes are the electrical activity, depolarization per se, contraction and intracellular [Ca2+] changes. To test the last three hypotheses, isolated bundles of denervated mouse soleus muscles were placed in narrow chambers (internal diameter 2 mm) which were periodically perfused with standard mixtures of Minimal Essential Medium (MEM) and calf serum. At 10-15-min intervals the chambers were filled for 15-43 s with a Ringer solution containing 55 microM ACh or 8-10 mM caffeine or with a solution containing 200 mM KCl. After 3-7 days the muscles were tested for ACh sensitivity by comparing the force developed in response to 110 microM ACh and that to 400 mM K methanesulphonate. In addition, the ACh receptor density was measured with 125I alpha-bungarotoxin. The results showed no difference in ACh sensitivity or receptor density between treated and untreated muscles. Small differences produced by caffeine were probably caused by muscle damage. Ca influx, but not efflux, was strongly elevated during exposure to ACh. It was concluded that neither depolarization per se nor contractile activity or the associated calcium movements have any effect on ACh receptors.