Neprilysin, obesity and the metabolic syndrome.

OBJECTIVE: Neprilysin (NEP), a zinc metalloendopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity, and investigated NEP production by adipocytes in-vitro. METHODS AND RESULTS: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects (P<0.01). Quantitative reverse transcriptase PCR (RT-PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg µl(-1), respectively; P<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice. CONCLUSION: In humans, NEP activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin-resistant mice. Our results indicate that NEP associates with cardiometabolic risk in the presence of insulin resistance and increases with obesity.

The influence of SLCO1B1 (OATP1B1) gene polymorphisms on response to statin therapy.

Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are well established in the treatment of hypercholesterolaemia and the prevention of coronary artery disease. Despite this, there is wide inter-individual variability in response to statin therapy, in terms of both lipid-lowering and adverse drug reactions. The major site of statin action is within hepatocytes and recent interest has focussed on genetic variation in hepatic influx and efflux transporters for their potential to explain these differences. In this review we explore current literature regarding the pharmacokinetic and pharmacodynamic influence of the common c.388A>G and c.521T>C single-nucleotide polymorphisms (SNPs) within the solute carrier organic anion transporter 1B1 (SLCO1B1) gene, encoding the organic anion transporter polypeptide 1B1 (OATP1B1) influx transporter. We discuss their potential to predict the efficacy of statin therapy and the likelihood that patients will experience adverse effects.

Functional expression of glucose-dependent insulinotropic polypeptide receptors is coupled to differentiation in a human adipocyte model.

OBJECTIVE: To establish that human adipocytes express functional glucose-dependent insulinotropic peptide (GIP) receptors and in particular the regulation of GIP receptor (GIPR) expression in the context of the dynamic process of adipocyte differentiation. DESIGN: A combination of semiquantitative real-time PCR and measurement of GIP-stimulated cAMP accumulation was used to establish the expression and functional coupling of GIPRs during in vitro differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes. RESULTS: Semiquantitative real-time PCR revealed that GIPR expression was substantially increased by day 4 of differentiation, reaching a maximum around 6-8 days (approximately 200-fold increase above undifferentiated cells, n=2). We also analysed the expression of the adipocyte fatty acid binding protein (FABP4) to relate GIPR expression to a molecular differentiation marker of adipogenesis. FABP4 expression was barely detectable in undifferentiated cells. However, following exposure to adipogenic medium, FABP4 expression gradually increased, with a maximal expression level around 10 days (approximately 1,600,000-fold increase above undifferentiated cells, n=2). Thus, the increases in GIPR mRNA during adipogenesis occur earlier than FABP4, suggesting that it might represent a gene expressed early in terminal differentiation and thus plays a role in fat droplet formation. A unit of 1 microM GIP failed to raise intracellular cAMP levels above basal levels in undifferentiated cells (n=3). In stark contrast, the 9-day differentiated cells produced a robust concentration-dependent increase in cAMP accumulation following stimulation with GIP, with an EC(50) value of 2.3 nM (n=3). The maximal response represented a 9-34-fold increase in cAMP accumulation above basal levels. CONCLUSIONS: This study demonstrates that GIPRs are expressed by human adipocytes, both GIPR mRNA and functional receptor expression being present in differentiated adipocytes but not in preadipocytes. Further investigation into the functional effects of GIP on differentiated SGBS cells could help towards understanding exactly how GIP regulates fat accumulation in human adipocytes.

The mechanism of angiotensin II-induced extracellular signal-regulated kinase-1/2 activation is independent of angiotensin AT(1A) receptor internalisation.

The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.

Effect of acute and chronic captopril infusion on blood pressure in the two-kidney, one clip hypertensive rat.

The effect on blood pressure (BP) of acute and chronic suppression of angiotensin (ANG) II was studied in the two-kidney, one clip hypertensive rat. Conscious, chronically catheterized rats were given a bolus injection of captopril (2.5 mg/kg) followed by a chronic infusion of either dextrose or captopril (1 mg/kg per h) lasting 5 days. Blood pressure was measured continuously by a computer technique. Following the acute injection of captopril, arterial BP fell from 165.1 +/- 19.4 mmHg (mean +/- s.d.) to a minimum of 137.6 +/- 23.3 mmHg after 15 min. Twelve hours after starting the chronic infusion of captopril, BP fell to a minimum of 112.5 +/- 19.4 mmHg. This was significantly lower than that after the acute injection of captopril. Blood pressure remained lower throughout the 5-day infusion ranging, on the 5th day, from 122.1 +/- 23.4 to 136.0 +/- 30.2 mmHg. In contrast, BP continued to rise in rats given dextrose chronically ranging, on the 5th day, from 163.6 +/- 23.8 to 180.4 +/- 22.5 mmHg. Both the fall in pressure after acute captopril and that after chronic captopril were related to pre-treatment levels of plasma renin concentration. These results suggest that in the two-kidney, one clip hypertensive rat ANG II, in addition to its acute vasoconstrictor property, contribute to the hypertension through a secondary effect, the mechanism of which is as yet uncertain.

Evidence of an important and direct role for protein kinase C in agonist-induced phosphorylation leading to desensitization of the angiotensin AT1A receptor.

1. The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT1 receptors remains unresolved. A major problem has been to isolate these receptors in a sufficiently purified form to allow study of their phosphorylation state. 2. A cleavable (His)6 affinity tag was introduced into the N-terminus of the recombinant AT1A receptor and stably expressed in human embryonic kidney cells. This affinity tag allowed rapid isolation, purification and determination of the phosphorylation state of the AT1A receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions. 3. Agonist-induced phosphorylation of the AT1A receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC specific inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase. 4. In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no effect on the level of desensitization observed. 5. These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT1 receptor desensitization.

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 x 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 x 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 x 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 x 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 x 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.

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.

Specific binding of atrial natriuretic peptide increases cyclic GMP levels in human astrocytoma cells.

Specific high-affinity binding sites (dissociation constant 100 pmol/l) for atrial natriuretic peptide (ANP) have been identified in the clone D384 derived from the human astrocytoma cell line G-CCM. Unrelated peptides such as angiotensin II, vasopressin and bradykinin did not compete for these sites. Of the atrial natriuretic peptides studied, both the human and rat ANP competed equally, while peptides with either C- or N-terminal residue missing or with no internal -S-S-bond either competed less effectively or did not compete at all. Human ANP stimulated the cells to increase their intracellular level of cyclic GMP in a time- and dose-dependent manner with maximum stimulation being approached but not reached at concentrations of 1 mumol/l. These results support both the notion that ANP has an important functional role within the brain and the concept of neurotransmitter/neuromodulator communication between neurones and glia.

Cultured mesenteric vascular smooth muscle cells express dopamine DA1-receptors.

Incubation of cultured mesenteric vascular smooth muscle cells with dopamine, in the presence of propranolol, caused an increase in cyclic AMP formation in a concentration-dependent manner (Ka apparent 6.8 +/- 0.5 microM). This effect of dopamine was inhibited by the DA1-receptor antagonist SCH 23390 (Ki = 1 nM). These results suggest that cultured mesenteric vascular smooth muscle cells express DA1-receptors linked to adenylate cyclase.

Thiol group identification at or near the agonist binding site of the vascular dopamine receptor.

Cultured mesenteric artery vascular smooth muscle cells derived from male Wistar rats, expressing both beta 2-adrenoceptors and dopamine DA1 receptors, were prelabelled for 2 h with [3H]adenine. [3H]cAMP formation stimulated by the addition of dopamine (plus propranolol 10 microM), isoprenaline or forskolin, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) was then determined. Exposure of cells to the thiol-oxidizing agent DTNB (5,5'-dithiobis-2-nitrobenzoic acid) following prelabelling, and prior to cAMP assay, resulted in a time-dependent inhibition of dopamine (0.1 mM)-induced cAMP formation, which obeyed the rules of first-order kinetics, being complete by 60 min. This inhibitory effect was observed to be dose related with 50% inhibition achieved at a concentration of 0.5 mM. Exposure to DTNB (5 mM) for 45 min abolished the cAMP response to dopamine (0.1 mM) with little effect on the response to forskolin (10 microM) or isoprenaline (10 microM). Prior addition of the dopamine DA1/D1 receptor selective partial agonist (+)-SKF 38393 (1 microM) preserved the dopamine induced cAMP formation despite DTNB exposure, while its stereo-enantiomer (-)-SKF 38393 (1 microM) protected only 25% of the response. Sequential exposure of cells to DTNB (5 mM) and then either vehicle or DTT (DL-dithiothreitol; 1 mM), each for 20 min periods, resulted in a 70% inhibition of dopamine induced cAMP formation which was almost completely reversed by the disulphide bridge cleaving compound DTT.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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)

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.

Inhibition of recombinant human cardiac L-type Ca2+ channel alpha1C subunits by 3-isobutyl-1-methylxanthine.

Inhibition of ion channels by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and related compounds has been demonstrated in various cell types, including the neuromuscular junction, GH3 cells and vascular smooth muscle cells. These effects may be unrelated to the actions of these compounds on cellular metabolism, intracellular Ca2+ stores and phosphodiesterase inhibition. In this study, the inhibition of recombinant human cardiac L-type Ca2+ channel alpha1C subunits by IBMX was examined using the whole-cell configuration of the patch clamp technique. Inhibition was repeatable, voltage-independent and associated with increased apparent channel inactivation. The actions of IBMX were unaffected in the presence of inhibitors of protein kinases A and G. The non-xanthine phosphodiesterase inhibitor rolipram had a small inhibitory effect on currents, but this was also unaffected by a protein kinase A inhibitor. These effects of IBMX could not be attributed to release of Ca2+ from intracellular stores. Our findings indicate that methylxanthines can inhibit the cardiac L-type Ca2+ channel alpha1C subunit in the absence of auxiliary subunits by an undetermined, possibly direct mechanism.

Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma x 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)

Functional domains of the C-terminus of the rat angiotensin AT1A receptor.

Previous work has shown that truncating the carboxyl terminus (C-terminus) of the rat angiotensin AT1A receptor to 309 amino acids abolished G-protein coupling and receptor internalization. This suggests that domains responsible for these functions lie beyond amino acid 309 of the C-terminus. The objective of this study was to determine the effect on angiotensin AT1A receptor function and regulation of deleting 41 amino acids from the C-terminus, which include the putative protein kinase C phosphorylation sites. Using site directed mutagenesis, the codon for Tyr319 was converted to a stop codon and the resulting truncated receptor permanently expressed in cultured human kidney cells. The properties of the truncated receptor were compared to those of the full length receptor. Expression of the truncated receptor was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of photolabelled membrane preparations. Angiotensin II activation of both full length and truncated receptors resulted in mobilization of inositol phosphates. However, whereas this was associated with rapid internalization of the full length receptor, the truncated receptor failed to internalize. Furthermore, pretreatment of cells with phorbol 12-myristate 13-acetate, a direct activator of protein kinase C, markedly attenuated the full length, but no the truncated receptor's ability to mobilise inositol phosphates. Thus, we conclude that the domain between amino acids 309 & 318 is important for G-protein coupling; that amino acids beyond 318 regulate internalization and one or more of the putative protein kinase C phosphorylation sites, present in the C-terminus of the angiotensin At1A receptor, actively regulate the receptor.

The effect of the angiotensin II (AT1A) receptor stably transfected into human neuroblastoma SH-SY5Y cells on noradrenaline release and changes in intracellular calcium.

A stable cell line expressing the angiotensin II (AII) receptor has been obtained by transfecting the human neuroblastoma SH-SY5Y with the plasmid pCEP4 containing the entire coding region of the rat angiotensin AII receptor AT1A. Angiotensin II (AII; 1-100 nM) evokes the release of [3H]noradrenaline ([3H]NA) in this cell line. Pretreatment with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances the AII-evoked release of [3H]NA approximately two-fold. Removal of extracellular Ca2+ ([Ca2+]o) decreases 100 nM AII-evoked release of [3H]NA by over 50% both in the presence and absence of TPA. AII increases intracellular Ca2+ ([Ca2+]i) in this cell line which is consistent with the AT1A receptor being coupled to phospholipase C. Pretreatment with 100 nM TPA for 8 min attenuated the effect of AII on [Ca2+]i. The effects of AT1A receptor stimulation are therefore regulated differently in this cell line by activation of protein kinase C (PKC). Thus a useful cell line has been obtained from the human neuroblastoma SH-SY5Y in which to study at the molecular level the mechanism(s) by which AII regulates NA release.

Identification of a B2-bradykinin receptor linked to phospholipase C and inhibition of dopamine stimulated cyclic AMP accumulation in the human astrocytoma cell line D384.

We have examined the activation of a phospholipase C signal transduction pathway by a B2-bradykinin receptor in the human astrocytoma cell line D384 and how this influences D1-dopamine receptor stimulated cyclic AMP accumulation. Addition of bradykinin to D384 cells resulted in a concentration-dependent (10(-11)-10(-6) M) increase in the accumulation of [3H]inositol phosphates and a similar concentration-dependent transient increase in specific [3H]beta-phorbol-12,13-dibutyrate binding which is indicative of translocation of protein kinase C from the cytosol to the membrane. Changes in intracellular Ca2+ of single cells, measured using the fluorescent indicator dye fura-2, indicated that bradykinin produced a rapid, but transient, increase in intracellular calcium. The Ca2+ response was largely independent of extracellular Ca2+ supporting the idea that receptor activation leads to mobilization of Ca2+ from intracellular stores. However, extracellular Ca2+ was required for a response to a rechallenge with bradykinin. The bradykinin B2-receptor agonist kallidin increased cytosolic Ca2+ in a similar manner to bradykinin. The Ca2+ response to bradykinin could be partially reduced in the presence of the B2-receptor antagonist [D-Arg0-Hyp,D-Phe7,beta-(2-Thienyl)-Ala5,8]-bradykinin, whereas the B1-receptor agonists (Des-Arg9]-bradykinin and [Des-Arg10]-kallidin were ineffective. Bradykinin was also found to attenuate dopamine stimulated cyclic AMP accumulation in D384 cells, at similar concentrations previously observed to stimulate the phospholipase C signal transduction pathway, in the presence of the phosphodiesterase inhibitor, rolipram. In contrast, no attenuation was observed in the presence of the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine, although the level of dopamine stimulated cyclic AMP observed was lower than in the presence of rolipram.(ABSTRACT TRUNCATED AT 250 WORDS)