Comparative potencies of 3,4-methylenedioxymethamphetamine (MDMA) analogues as inhibitors of [3H]noradrenaline and [3H]5-HT transport in mammalian cell lines.

BACKGROUND AND PURPOSE: Illegal 'ecstasy' tablets frequently contain 3,4-methylenedioxymethamphetamine (MDMA)-like compounds of unknown pharmacological activity. Since monoamine transporters are one of the primary targets of MDMA action in the brain, a number of MDMA analogues have been tested for their ability to inhibit [3H]noradrenaline uptake into rat PC12 cells expressing the noradrenaline transporter (NET) and [3H]5-HT uptake into HEK293 cells stably transfected with the 5-HT transporter (SERT). EXPERIMENTAL APPROACH: Concentration-response curves for the following compounds at both NET and SERT were determined under saturating substrate conditions: 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxy-N-hydroxyamphetamine (MDOH), 2,5-dimethoxy-4-bromophenylethylamine (2CB), 3,4-dimethoxymethamphetamine (DMMA), 3,4-methylenedioxyphenyl-2-butanamine (BDB), 3,4-methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) and 2,3-methylenedioxymethamphetamine (2,3-MDMA). KEY RESULTS: 2,3-MDMA was significantly less potent than MDMA at SERT, but equipotent with MDMA at NET. 2CB and BDB were both significantly less potent than MDMA at NET, but equipotent with MDMA at SERT. MBDB, DMMA, MDOH and the MDMA metabolites HMA and HMMA, were all significantly less potent than MDMA at both NET and SERT. CONCLUSIONS AND IMPLICATIONS: This study provides an important insight into the structural requirements of MDMA analogue affinity at both NET and SERT. It is anticipated that these results will facilitate understanding of the likely pharmacological actions of structural analogues of MDMA.

Novel "plum pudding" gels as potential drug-eluting stent coatings: controlled release of fluvastatin.

This study evaluated novel structural motifs known as "plum pudding" gels as potential drug-eluting stent coatings. Controlled delivery of a HMG-CoA reductase inhibitor (statin) from the intravascular stent surface represents a potential therapeutic modality for prevention of in-stent restenosis (ISR). In this study, gels were comprised of fluvastatin-loaded thermoresponsive microgel particles containing the relatively hydrophilic N-isopropylacrylamide (NiPAAm), mixed with the more hydrophobic N-tert-butylacrylamide (NtBAAm) in different wt/wt ratios: 85/15, 65/35, and 50/50, randomly dispersed in a 65/35 or 85/15 NiPAAm/NtBAAm copolymer matrix. Fluvastatin release from 5 microm copolymer films was greatest from the most hydrophilic systems and least from the more hydrophobic systems. Release from hydrophobic matrices appeared to be via Fickian diffusion, enabling use of the Stokes-Einstein equation to determine diffusion coefficients. Release from hydrophilic matrices was non-Fickian. Eluted drug retained its bioactivity, assessed as selective inhibition of human coronary artery smooth muscle cell proliferation. When stainless steel stent wires were coated (25 microm thickness) with fluvastatin-loaded 65/35 microgels in an 85/15 copolymer matrix, drug elution into static and perfused flow environments followed similar elution profiles. In contrast to elution from copolymer films cast on flat surfaces, diffusion from stent wires coated with hydrophilic and hydrophobic systems both followed Fickian patterns, with slightly larger diffusion coefficients for elution from the flow system. We conclude that manipulation of the relative hydrophobicities of both microgel and matrix components of "plum pudding" gels results in tightly regulated release of fluvastatin over an extended time period relevant to initiation and propagation of ISR.

Blockade of noradrenaline transport abolishes 4-methylthioamphetamine-induced contraction of the rat aorta in vitro.

The aim of this study was to characterize the effects of 4-methylthioamphetamine (4-MTA) on contractility and noradrenaline (NA) transport and release in the isolated rat aorta. Descending thoracic aortic rings were isolated from male Wistar rats (220-240 g) and the effect of 4-MTA on contractility was measured by isometric force displacement. 4-MTA (0.1 microm-1 mm) induced a concentration-dependent contraction of aortic rings, with a pD(2) of 4.40 +/- 0.38, and an E(max) of 0.80 +/- 0.05 g tension. The alpha(1)-adrenoceptor antagonist, prazosin (1 microm) and alpha(2) antagonist, yohimbine (1 microm) inhibited maximal contraction to 100 microm 4-MTA by 45.0 +/- 6.7% and 53.5 +/- 7.1% of control values respectively, whereas the 5-hydroxytryptamine (5-HT) antagonist, ketanserin (100 nm) had no effect on the 4-MTA-mediated contraction. The specific NA transport inhibitor, nisoxetine (1 microm) abolished contraction of the aorta by 4-MTA. 4 Nisoxetine-sensitive [(3)H]-NA transport in aortic rings was measured over a concentration range of 0-5 microm [(3)H]-NA, and had a maximal rate of transport (V(max)) of 0.77 +/- 0.07 pmol [(3)H]-NA min(-1) mg(-1) protein and a Michaelis affinity constant (K(M)) of 2.3 +/- 0.5 microm. 4-MTA inhibited nisoxetine-sensitive [(3)H]-NA transport with a pIC(50) of 6.16 +/- 0.18 and the pIC(50) for inhibition of nisoxetine-sensitive [(3)H]-NA transport by 3,4-methylenedioxymethamphetamine (MDMA) was 6.83 +/- 0.13. 4-MTA (1-100 microm) significantly stimulated release of pre-loaded [(3)H]-NA from aortic rings and 4-MTA-induced [(3)H]-NA release was inhibited by 1 microm nisoxetine. These data suggest that 4-MTA causes contraction of the rat aorta in vitro by a mechanism that is consistent with an ability to cause release of NA at the level of the NA transporter. It is concluded that 4-MTA has the potential to increase the extracellular concentration of NA peripherally as well as centrally, and that this may cause adverse cardiovascular effects in its users.

The beta-adrenoceptor is precoupled to Gs in chicken erythrocyte membranes.

In this study we seek to elucidate the mechanism of hormone-independent adenylate cyclase stimulation by Gpp(NH)p in chicken erythrocyte membranes, and the inhibition of this stimulation by propranolol. Membrane treatment with isoprenaline + GMP increased Gpp(NH)p stimulation to near maximal levels [obtainable with isoprenaline + Gpp(NH)p], but reduced stimulation by NaF. The stimulation by Gpp(NH)p was stereoselectively inhibited by propranolol, but not by equal concentrations of the local anaesthetic lignocaine. Propranolol's inhibitory action was abolished following membrane treatment with isoprenaline/GMP. In contrast to its inhibition of Gpp(NH)p stimulation, propranolol did not alter Gpp(NH)p-mediated 3H-GDP release from membranes. The polyene antibiotic filipin, which uncouples receptor (R) from Gs, also abolished Gpp(NH)p stimulation and this effect was partly overcome by membrane treatment. These results are consistent with a model in which free R exists in equilibrium with precoupled R.Gs complexes in the absence of hormone. These complexes are activated by Gpp(NH)p and dissociated by antagonists. The existence of such complexes is a prerequisite for Gpp(NH)p stimulatory action.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes.

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein Gs linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of Gi linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [125I]cyanopindolol to membranes, suggesting that catecholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G alpha i2, G alpha q, and G beta 1. In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AIF4-, GTP[S], or purified G beta gamma subunits. Finally, no specific binding to membranes of the alpha 1-adrenoceptor ligand [3H]prazosin or the alpha 2-adrenoceptor ligand [3H]yohimbine was obtained. In conclusion, this study provides evidence for a Gs-dependent stimulation of AC, and for the presence of Gi2 and Gq/11, which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors.

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.

Cytotoxicity-associated effects of reactive oxygen species on endothelin-1 secretion by pulmonary endothelial cells.

In this study bovine pulmonary artery endothelial cells (BPAEC) were used as a model system to investigate the effects of the hypoxanthine-xanthine oxidase (HXXO) oxygen radical donor system on ET-1 secretion into pulmonary vasculature. Incubation of BPAEC with HXXO for 4 h caused a significant reduction in ET-1 secretion, which was significantly offset by allopurinol or catalase, but not by Cu/Zn superoxide dismutase (SOD). ET-1 secretion was also reduced by H2O2, and this effect was again significantly offset by catalase. XO alone also reduced ET-1 secretion, but to a significantly lesser degree than did HXXO, and this effect was not offset by allopurinol, catalase, or SOD. None of the oxidant treatments were associated with a loss of immunoreactive ET-1 from endothelial cell medium containing synthetic peptide. The HXXO- and H2O2-mediated reductions in ET-1 secretion were accompanied by evidence of reduced cell viability. This loss of viability was absent when cells were treated with HXXO + catalase, allopurinol, or mercaptopropionyl glycine, but not when SOD was present. We conclude that under conditions of oxidative stress, the pulmonary vascular endothelium responds by secreting less ET-1. This may be relevant to its vasodilator functions in the pulmonary vasculature, which would therefore be compromised when the endothelium is exposed to oxidant stress.

Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery.

In this study specific high affinity binding sites for atrial natriuretic factor (rANF(99-126] have been identified on cultured endothelial cells of bovine pulmonary artery origin (BPAEC). A time-dependent rise in cellular cGMP levels stimulated by rANF(99-126) was followed by release of the nucleotide into the incubation medium. The use of truncated, ring-deleted and linear atrial peptide analogs in competitive displacement analysis and measurement of cGMP accumulation indicated that only a minor proportion (5-11%) of the available receptor pool was of the ANF-B receptor subtype, linked to guanylate cyclase, with the remaining major proportion possibly of the ANF-C (clearance) receptor subtype. The existence of two ANF receptor subtypes in this cell culture model would suggest a significant role for the circulating peptide in modulation of pulmonary endothelial cell function, which would influence or complement its direct actions on the underlying vasculature of the pulmonary circulation.

Unimpaired coupling of phosphorylated, desensitized beta-adrenoceptor to Gs in a reconstitution system.

Heterologous desensitization of turkey erythrocyte beta-adrenoceptors correlates with receptor phosphorylation and impaired receptor-Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837-840]. We have purified beta-receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor-Gs coupling was assessed by measuring hormone-dependent Gs activation by GTP gamma S and GTPase activity. While in membranes prepared from desensitized cells, receptor-Gs coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.

Adenylyl cyclase activity in clonally derived human myoblast cultures: evidence for myoblast heterogeneity.

In vitro myogenesis recapitulates the programme of myogenesis in vivo. During the process of muscle differentiation, cAMP plays an important role in the control of gene expression and in the integration of metabolic functions. cAMP generation may be affected by drugs or hormones that interact with the membrane-bound enzyme adenylyl cyclase, including adrenergic agents and glucocorticoids. In this study, adenylyl cyclase activity was evaluated in membranes prepared from human clonally derived muscle cultures. In control cultures, there was considerable inter-clonal variation in basal, sodium-fluoride and forskolin-stimulated adenylyl cyclase activity. Cultures differed in their response to steroids: adenylyl cyclase activity was markedly enhanced in some clones, and was significantly inhibited in other clones. Pre-treatment of cultures with pertussis toxin indicated that the effects of steroids are mediated in part by modulation of G-protein activity. These findings indicate a substantial heterogeneity among myoblast clones with respect to the modulating effect of steroids on adenylyl cyclase activity. This observation may account for the conflicting reports of steroid effects on muscle in vitro, and may be of relevance to the understanding of possible transmembrane signalling alterations in muscle disease.

Differential effects of D600, nifedipine and dantrolene sodium on excitation-secretion coupling and presynaptic beta-adrenoceptor responses in rat atria.

The stimulation-evoked release of tritium was measured from rat atria labelled with [3H]-noradrenaline. The calcium dependence of evoked release and the facilitation of this release via activation of presynaptic beta-adrenoceptors were examined using D600 (methoxyverapamil), nifedipine and dantrolene sodium. Both D600 and nifedipine at dose levels of 20 and 100 microM inhibited evoked release. Dantrolene (20, 100 microM) reduced release by 25%, the effect being maximal at 20 microM. In the presence of 20 nM isoprenaline, a facilitation of evoked release occurred, which was blocked by 0.1 microM (-)-propranolol. The facilitatory action of isoprenaline was abolished by omission of calcium from the buffer, or by D600 or nifedipine, (100 microM). In contrast, the response to isoprenaline was not modified by dantrolene (20, 100 microM). It is concluded that the evoked release of noradrenaline (NA) utilizes Ca from both intra- and extracellular sources and that isoprenaline increases NA secretion by promoting the depolarization-induced influx of Ca.

Inhibition by fatty acids of cyclic AMP-dependent protein kinase activity in brush border membranes isolated from human placental vesicles.

1. The inhibitory effects of arachidonic acid (AA) and a number of structurally related fatty acids on cyclic AMP-dependent protein kinase activity have been investigated in brush border membranes (BBM) prepared from human placental vesicles. 2. BBM vesicles were characterized by electron microscopy and displayed enrichment of the appropriate marker enzymes, alkaline phosphatase and gamma-glutamyltranspeptidase; BBM were prepared by vesicles lysis in hypotonic medium. 3. Cyclic AMP-dependent protein kinase (PKA) activity was measured in BBM. At 1 microM, cyclic AMP stimulated a 4.2 +/- 0.06 fold increase over basal levels of [32P]-phosphate incorporation into the synthetic substrate kemptide and this effect was abolished by a selective PKA inhibitor. By use of synergistic pairs of site-selective cyclic AMP analogues, the kinase was identified as the type II enzyme. 4. Cyclic AMP-stimulated PKA activity was inhibited by 10 microM AA and this effect was significantly enhanced by nordihydroguaiaretic acid (NDGA) + indomethacin (Indo), inhibitors of the lipoxygenase and cyclo-oxygenase pathways of AA metabolism respectively. 5. Oleic acid, elaidic acid, but not caprylic or palmitic acids, also significantly inhibited PKA activity and this effect was again enhanced by NDGA + Indo. While arachidonyl alcohol alone was not inhibitory, in the presence of the metabolic inhibitors a significant reduction in stimulated activity was observed. 6. The commercially available PKA type II holoenzyme (activated by cyclic AMP), but not the free catalytic subunit, was inhibitable by AA, oleic or elaidic acids. 7. These results suggest that PKA localized to the brush border membrane of human placental vesicles is inhibited by fatty acids which may compete with cyclic AMP for binding to the kinase regulatory subunit. The reported inhibition by fatty acids of cyclic AMP-dependent Cl- secretion in epithelial cells may therefore be due in part to negative regulation of a Cl- channel-associated PKA.

In vitro neuronal and vascular responses to 5-HT in rats chronically exposed to MDMA.

1. This study examined the effects of chronic exposure of rats to 3,4-methylenedioxymethamphetamine (MDMA) on [(3)H]5-hydroxytryptamine ([(3)H]5-HT) re-uptake into purified rat brain synaptosomes, 5-HT-induced isometric contraction of aortic rings and [(3)H]5-HT re-uptake into rat aorta. 2. Rats were administered MDMA (20 mg kg(-1) i.p.) twice daily over 4 days. One, 7, 14 or 21 days post treatment, whole brain synaptosomes and descending thoracic aortic rings were prepared for investigation. 3. Chronic MDMA treatment significantly reduced the maximum rate (V(max)) of specific high-affinity [(3)H]5-HT re-uptake 1 day after treatment and for up to 21 days post-final administration of MDMA. Direct application of MDMA (100 microM) abolished synaptosomal re-uptake of [(3)H]5-HT in vitro. 4. Chronic MDMA administration significantly reduced the maximum contraction (E(max)) to 5-HT at 1 and 7 days after treatment, but not at 14 or 21 days. 5. Chronic MDMA administration had no effect on sodium-dependent [(3)H]5-HT re-uptake into aorta 1 day after treatment, nor did 100 microM MDMA have any direct effect on [(3)H]5-HT uptake into aortic rings in vitro. 6. These results show, for the first time, an altered responsiveness of vascular tissue to MDMA after chronic administration. In addition, they demonstrate a difference in the sensitivity of central and peripheral 5-HT uptake systems to chronic MDMA exposure, and suggest that the action of MDMA in the cardiovascular system does not arise from a direct effect of MDMA on peripheral 5-HT transport.

Sequestered A431 cell beta-adrenoceptors remain functional after homologous desensitization.

This study seeks to investigate the functional state of sequestered beta-adrenoceptors in A431 cells which have undergone homologous desensitization. Incubation of cells with isoprenaline under desensitizing conditions caused a reduction (a) in the number of cell surface beta-receptors as measured by 3H-CGP 12,177 binding, and (b) in the extent of agonist-stimulatable adenylate cyclase activity in membranes prepared from desensitized cells. We infer that those receptors have been sequestered to an internal membrane site since they were detectable by the lipophilic ligand 125I-CYP but not by the more hydrophilic 3H-CGP 12,177. We confirmed that sequestration had occurred by fractionation on non-linear sucrose density gradients of membranes prepared from desensitized cells. The lighter density membrane fraction contained up to 50% of the total receptor pool after desensitization, but only 5-7% of membrane (fluoride-stimulatable) adenylate cyclase. Fusion of desensitized cells in the presence of polyethylene glycol caused a reassociation of sequestered beta-receptors with their biochemical effector Gs since agonist-dependent adenylate cyclase stimulation was restored to the levels measurable after fusion of non-desensitized cells. We conclude that sequestered beta-receptors in desensitized A431 cells are fully functional.

Evidence for modulation of hydrogen peroxide-induced endothelial barrier dysfunction by nitric oxide in vitro.

Acute effects of the nitric oxide (NO) donors sodium nitroprusside and glyceryl trinitrate on hydrogen peroxide (H2O2)-induced increases in endothelial monolayer permeability to trypan blue-labelled bovine serum albumin have been investigated in vitro. Exposure of bovine pulmonary artery endothelial cell monolayers to 0.2 mM H2O2 for 20 min caused a significant increase in percentage trypan blue-labelled albumin transfer from the lumenal to the ablumenal compartment (basal 6.0 +/- 0.6 to 25.4 +/- 0.9%, n = 4, P < 0.0005). In separate experiments 100 microM sodium nitroprusside significantly enhanced the effect of 0.2 mM H2O2 (from 7.4 +/- 1.4 to 11.9 +/- 1.5%, n = 9, P < 0.0001) but did not alter albumin transfer in the absence of H2O2. This additive effect appeared to be due to NO release from sodium nitroprusside, since nitrite concentration in the medium overlying cells treated with 100 microM sodium nitroprusside was 19.9 +/- 1.8 microM (n = 12). Significantly less nitrite (3.5 +/- 0.5 microM, n = 12, P < 0.0001) was found in the medium overlying cells treated with 100 microM glyceryl trinitrate, which in contrast to sodium nitroprusside, inhibited the permeability increase caused by H2O2 (from 15.6 +/- 3.3 to 13.8 +/- 3.1%, n = 6, P < 0.001). Furthermore 10 microM sodium nitroprusside, which released comparable amount of nitrite (4.5 +/- 0.4 microM, n = 6) to 100 microM glyceryl trinitrate, also inhibited the permeability increase caused by H2O2 (from 20.7 +/- 0.4 to 19.4 +/- 0.3%, n = 9, P < 0.01). We conclude that relatively large amounts of NO released from 100 microM sodium nitroprusside exacerbate the barrier dysfunction caused by H2O2, while lower amounts of NO give a small amount of cytoprotection.

Evidence for signalling by big endothelin-1 via conversion to endothelin-1 in pulmonary artery smooth muscle cells.

In this study, signalling by big endothelin-1 (big ET-1) and a role for the phosphoramidon-sensitive endothelin converting enzyme in this signalling was investigated in smooth muscle cells cultured from bovine pulmonary artery. In whole cells, both ET-1 and big ET-1 stimulated inositol phosphate accumulation in a time- and concentration-dependent manner. The endothelin ET-A receptor antagonist (BQ 123, 1 microM) significantly inhibited stimulation by ET-1 (10 nM) or big ET-1 (100 nM). When cells were pretreated with 100 microM phosphoramidon, the stimulation by big ET-1, but not ET-1 was abolished. In separate experiments, when cells were incubated with exogenous big ET-1, a time-dependent phosphoramidon-sensitive conversion to ET-1 was detected by radioimmunoassay. These results are consistent with the presence of a phosphoramidon-sensitive endothelin converting enzyme on the surface of bovine pulmonary artery smooth muscle cells, which may play a role in regulating signalling by circulating big ET-1.

Effects of the ANF-C receptor ligand des[Cys105,Cys121]rANF(104-126) on ANF internalization and cGMP production by bovine pulmonary artery endothelial cells.

A role for the ANF-C receptor ligand des[Cys105,Cys121]rANF(104-126) in ANF receptor-effector coupling was investigated using cultured bovine pulmonary artery endothelial cells as a model system. The ligand was equipotent with rANF(99-126) in displacement of [125I]des[Cys105,Cys121]rANF(104-126) binding to whole cells and labelled only one population of ANF receptors as shown by affinity crosslinking experiments. In cells pretreated with des[Cys105, Cys121]rANF(104-126), internalization of [125I]rANF(99-126) binding was reduced and enhanced accumulation of cGMP was observed under basal conditions, or in the presence of a low concentration (0.1 nM) of rANF(99-126). These results provide a mechanistic basis for the observed enhancement of ANF-dependent vasodilation by ANF-C receptor selective ligands.

The ANF-C receptor is not linked to adenylyl cyclase inhibition in bovine pulmonary artery endothelial cells.

The possible inhibition of adenylyl cyclase activity by atrial peptides selective for the ANF-C receptor was investigated in bovine pulmonary artery endothelial cells. In these cells isoprenaline, guanine nucleotide and forskolin dose-dependently increased activity over basal levels. In the presence of rANF(99-126), these dose-dependent increases were not reduced, nor were they affected by the ANF-C receptor selective analogue C-ANF(102-121). Furthermore, the selective analogues rANF(103-123) and des[Cys105,Cys121]rANF104-126 had no effect on basal or stimulated adenylyl cyclase activity. It can be concluded that ANF-C receptors are not linked to inhibition of adenylyl cyclase in these cells.

Extended delivery of the antimitotic agent colchicine from thermoresponsive N-isopropylacrylamide-based copolymer films to human vascular smooth muscle cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of an antimitotic agent to human vascular smooth muscle cells (HASMC) over an extended period of time. Copolymer films were prepared using a 50:50 (w/w) ratio of N-isopropylacrylamide (NiPAAm) monomer to the more hydrophobic N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (0.1 micromol per film) at room temperature. Colchicine release from films was sustained over a 14-day period. At 24 h postloading, the concentration of colchicine in the medium overlying films was 2.12 +/- 0.16 microM; this fell to 0.20 +/- 0.01 microM at 7 days and decreased further to 0.12 +/- 0.01 microM after 14 days. Colchicine released from copolymer films inhibited proliferation when subsequently placed on HASMC: at 0.1 microM, released colchicine reduced proliferation to 18.5 +/- 0.8% of control cells (p < 0.001, n = 9). The antiproliferative effect of released colchicine was comparable to that of native colchicine, as observed in separate experiments. Furthermore, colchicine released from 50:50 polymer films inhibited the proliferation of cells grown in the same environment as the copolymer. Inhibition of cell proliferation was not due to the release of cytotoxic particles from the copolymer because medium incubated with copolymer for 5 days and then applied to HASMC did not alter cell viability. In conclusion, this study demonstrates that 50:50 NiPAAm:NtBAAm copolymers can deliver bioactive concentrations of the antimitotic agent colchicine to human vascular cells over an extended period of time.

Plasma atrial natriuretic peptide is elevated in patients with hypertrophic cardiomyopathy.

STUDY OBJECTIVES: To determine if plasma levels of atrial natriuretic peptide are elevated in patients with hypertrophic cardiomyopathy and to determine the relationship of atrial natriuretic peptide to symptoms and echocardiographic indices of left ventricular structure and diastolic function in these patients. DESIGN: A prospective study in which atrial natriuretic peptide was measured in peripheral venous plasma in 14 patients (age 44 +/- 14 years) with hypertrophic cardiomyopathy and 17 healthy controls. Echocardiography was performed in all cases and 30 controls to examine indices of left heart structure and function. All patients underwent clinical evaluation. RESULTS: The concentration of atrial natriuretic peptide was significantly higher in patients with hypertrophic cardiomyopathy than controls, (17.86 +/- 8.72 vs. 6.22 +/- 3.26 pmol/l, P = 0.0001). Diastolic dysfunction was observed in 11 of 14 patients with hypertrophic cardiomyopathy. No correlation was demonstrated between atrial natriuretic peptide levels and the degree of diastolic dysfunction, septal or free wall thickness, left atrial size, degree of mitral regurgitation or New York Heart Association functional class. CONCLUSIONS: Plasma levels of atrial natriuretic peptide are elevated in patients with hypertrophic cardiomyopathy but do not correlate with symptoms or echocardiographically-derived indices of left ventricular structure or diastolic function.