Susan E. Bryson

Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma × glioma hybrid, NG-108-15

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([125I]angiotensin II), in undifferentiated cells, to a Bmax of 1077 (1070-1268) fmol/mg in 5-day-differentiated cells. The affinity (Kd) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5-10) nM. The recently available selective non-peptide antagonists, DuP 753 and PD 123177 and the peptide analogues of angiotensin II, CGP 42112A and p-aminophenylalanine6 angiotensin II, were used to characterize the angiotensin II receptors by competing for 125I-[Sar1-Ile8]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT1 and after differentiation AT2. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4-5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca2+ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT1 antagonist, DuP 753, indicating an action at the AT1 receptor subtype in both undifferentiated and differentiated cells. The selective AT2 antagonist, PD 123177 was without effect on the angiotensin II induced increase in intracellular Ca2+. This effect of DuP 753 on Ca2+ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative pharmacology of recombinant rat AT1A, AT1B and human AT1 receptors expressed by transfected COS-M6 cells.

1 Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes. 2 We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non‐peptide AT1 receptor antagonist. Using a radioligand‐receptor binding assay and membranes prepared from COS‐M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes. 3 The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]‐Sar1Ile8 angiotensin II for binding to the AT1 receptors. 4 Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences. 5 It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of the dopamine receptor coupled to cyclic AMP formation expressed by rat mesenteric artery vascular smooth muscle cells in culture.

1 Mesenteric artery vascular smooth muscle cells derived from male Wistar rats and grown in culture were prelabelled with [3H]‐adenine and exposed to a range of dopamine receptor agonists and antagonists. Resultant [3H]‐cyclic AMP formation was determined and concentration‐effect curves constructed, in the presence of propranolol (10−6 m) and the phosphodiesterase inhibitor IBMX (5× 10−4 m). 2 Ka apparent values for D1/DA1 dopamine receptor agonists SKF 38393, fenoldopam, 6,7‐ADTN, and dopamine were 0.06, 0.59, 4.06 and 5.77 × 10−6 m respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine of approximately 48% and 24% respectively. 6,7‐ADTN, in contrast, behaved as a full agonist. 3 Dopamine‐stimulated cyclic AMP formation was inhibited in a concentration‐dependent manner by the D1/DA1 dopamine receptor selective antagonists, SCH 23390 and cis‐flupenthixol (Ki values 0.53 and 36.1 × 10−1 m respectively). In contrast, the D2/DA2 dopamine receptor selective antagonists, domperidone and (−)‐sulpiride, were less potent (Ki values 2.06 and 5.82 × 10−6 m respectively). Furthermore, the stereoisomers of SCH 23390 and cis‐flupenthixol, SCH 23388 and trans‐flupenthixol, were at least two orders of magnitude less potent (Ki values 0.14 and 13.2 × 10−6 m respectively) indicating the stereoselective nature of this receptor. 4 Our results indicate that rat mesenteric artery vascular smooth muscle cells in culture express a dopamine receptor coupled to cyclic AMP formation, which has the pharmacological profile, characteristic of the D1 dopamine receptor subfamily.

Characterization of the dopamine receptor expressed by rat glomerular mesangial cells in culture

Incubation of cultured rat glomerular mesangial cells with dopamine caused an increase in cyclic AMP formation in a concentration-dependent manner (Ka apparent 2.2 microM). The selective dopamine D1 receptor agonists, fenoldopam, SKF 38393 and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) also produced concentration-dependent increases in cyclic AMP with mean Ka apparent values of 0.04 microM, 0.02 microM and 1.02 microM, respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine, of approximately 60 and 35%, respectively. The dopamine analogue, 6,7-ADTN, in contrast, behaved as a full agonist. Dopamine-stimulated cAMP formation was inhibited in a concentration-dependent manner by the D1-selective antagonist, SCH 23390, with a Ki of 0.06 nM. In contrast, the D2-selective antagonist, domperidone, was four orders of magnitude less potent than SCH 23390, having a Ki of 2072 nM. In addition, SCH 23388, the stereoisomer of SCH 23390, was observed to be two orders of magnitude less potent than SCH 23390, indicating the stereoselective nature of the receptor. The potency series for the selective agonists and antagonists is the same as that described, using identical experimental conditions, for the D1 receptor expressed by a cell line of central origin confirming that the peripheral DA1 and the central D1 dopamine receptor are pharmacologically similar.

Glucocorticoids regulate the expression of angiotensin AT1 receptors, in the human hepatoma cell line, PLC-PRF-5

The effects of different steroids on the expression of angiotensin AT1 receptors by the human hepatoma cell line, PLC-PRF-5 was studied. Dexamethasone and aldosterone decreased the specific binding of [3H]angiotensin II to intact PLC-PRF-5 cells by 57 +/- 4% and 54 +/- 2%, respectively, compared to control, untreated cells. EC50 values for dexamethasone, cortisol and aldosterone were 1.8 +/- 0.6, 40 +/- 6, and 310 +/- 20 nM, respectively, suggesting that these effects were mediated via a glucocorticoid receptor. Scatchard analysis revealed that dexamethasone decreased the number of angiotensin AT1 receptors expressed (50 +/- 4% relative to control) with no change in receptor affinity. Treating cells with dexamethasone in the presence of either an angiotensin converting enzyme inhibitor or an angiotensin II receptor antagonist did not prevent the reduction in angiotensin AT1 receptor expression, ruling out a mechanism involving a dexamethasone induced increase in endogenous angiotensin II production. A ribonuclease protection assay established that the steady state level of angiotensin AT1 receptor mRNA in dexamethasone treated cells was reduced to 34.7 +/- 8.4% of untreated cells. The decrease in the number of angiotensin AT1 receptors expressed on the cell surface after treatment with dexamethasone therefore seems likely to reflect the decreased steady state level of the mRNA coding for this receptor.

Phenoxybenzamine mediated inhibition of the vascular dopamine D1 receptor

Cultures of rat mesenteric artery vascular smooth muscle cells express both vascular dopamine D1 receptors and beta 2-adrenoceptors but not alpha 1- or alpha 2-adrenoceptors, permitting direct investigation of the dopamine D1 receptor antagonist activity of phenoxybenzamine. After incubating cells with phenoxybenzamine (10(-5) M) for 20 min, an 80% inhibition of dopamine-induced (10(-4) M) cAMP formation was observed. Isoprenaline-induced (10(-5) M) cAMP formation remained unaffected by phenoxybenzamine. Inhibition of the dopamine response following 20 min incubation with phenoxybenzamine, was concentration-related and could not be reversed by repeated washing. Mean IC50 (95% confidence limits) = 4.68 x 10(-6) M (3.86-5.01). Exposure of cells to the selective dopamine D1 receptor partial agonist (+)-SKF 38393 (10(-6) M) prior to phenoxybenzamine incubation, resulted in protection of dopamine-induced cAMP formation. Exposure of cells to the stereo-enantiomer (-)-SKF 38393 (10(-6) M) did not produce any protective effect. The concentration-effect curve for (+)-SKF 38393 mediated protection had a mean EC50 value of 0.11 x 10(-6) M (0.10-0.11), which is comparable with the Ka apparent value (0.06 x 10(-6) M) for this compound when acting as an agonist to induce cAMP formation via the vascular dopamine D1 receptor. Previous studies of the vascular dopamine D1 receptor are likely to have been influenced by the frequent use of phenoxybenzamine, which we have shown to act as a potent antagonist at this site.