Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling.

Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and ß-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different ß-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Glycosaminoglycan synthesis and structure as targets for the prevention of calcific aortic valve disease.

Calcific aortic valve disease is frequently driven by ageing and the obesity-associated metabolic syndrome, and the increasing impact of these factors indicates that valve disease will become a cardiovascular disease of considerable significance. This disease is now thought to be an active cell-based disease process, which may therefore be amenable to therapeutic intervention. Some similarities are apparent with atherosclerosis. The accumulation of lipid, possibly by retention by proteoglycans and the attraction of inflammatory cells by hyaluronan, may be common to the early stages of both pathologies. The synthesis and structure of glycosaminoglycans, proteoglycans, and hyaluronan are exquisitely regulated, and the signalling pathways controlling these processes may provide tissue-specific opportunities for concomitant prevention of atherosclerosis and calcific aortic valve disease.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding.

AIMS/HYPOTHESIS: Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. METHODS: Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 micromol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [(35)S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. RESULTS: Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8+/-12.4 microg/ml to 77.7+/-17 microg/ml under basal conditions, from 24.9+/-4.6 microg/ml to 39.1+/-6.1 microg/ml in the presence of TGF-beta1, and from 9.5+/-4.4 microg/ml to 31.1+/-3.4 microg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-beta1 inhibited the incorporation of [(35)S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. CONCLUSIONS/INTERPRETATION: Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Preclinical studies of opioids and opioid antagonists on gastrointestinal function.

Opioid receptors in the gastrointestinal (GI) tract mediate the effects of endogenous opioid peptides and exogenously administered opioid analgesics, on a variety of physiological functions associated with motility, secretion and visceral pain. The studies reviewed or reported here describe a range of in vivo activities of opioid receptor antagonists upon GI function in rodents, focusing on mu receptors. Naloxone, and the peripherally acting mu-opioid receptor antagonists alvimopan and methylnaltrexone, reverse morphine-induced inhibition of GI transit in mice and rats, and morphine- or loperamide-induced inhibition of castor oil-induced diarrhoea in mice. At doses producing maximal reversal of morphine-induced effects upon GI transit, only the central nervous system (CNS) penetrant antagonist naloxone was able to reverse morphine-induced analgesia. Both central and peripheral opioid antagonists may affect GI function and/or visceromotor sensitivity in the absence of exogenous opioid analgesics, suggesting a constitutive role for endogenous opioid peptides in the control of GI physiology. Furthermore, in contrast to naloxone, alvimopan does not produce hypersensitivity to the visceromotor response induced by nociceptive levels of colorectal distension in a rodent model of post-inflammatory colonic hypersensitivity, suggesting that in the periphery endogenous mu-opioid receptor-mediated mechanisms do not regulate colonic sensitivity. The data support the hypothesis that peripherally acting opioid antagonists may be able to selectively block opioid receptors in the GI tract, thereby preserving normal GI physiology, while not blocking the effects of endogenous opioid peptides or exogenous opioid analgesics in the CNS. These findings suggest that the primary sites of action of mu-opioid agonists with respect to inhibition of GI function are in the periphery, whereas analgesic activity resides primarily in the CNS.

Regulation of glycosaminoglycan structure and atherogenesis.

Cardiovascular disease is the major cause of premature death in modern society, and its impact is increasing due to rising rates of obesity and type 2 diabetes. Clinical studies based on targeting metabolic abnormalities and biomarkers demonstrate significant benefits, but always an element of disease remains which is resistant to treatment. Recent evidence has strongly implicated an early interaction of atherogenic lipoproteins with vascular matrix proteoglycans as the initiating step in atherogenesis. Expert commentary has pointed to the need for vascular directed therapies to provide reductions in the residual disease component. We propose that the regulation of synthesis and thus structure of glycosaminoglycans on proteoglycans provides a potential pathway to this reduction. We review existing evidence that the vascular synthesis of glycosaminoglycan chains can be regulated in a manner which reduces lipoprotein binding and the potential application of this strategy to attenuation of the current cardiovascular disease pandemic.

Thiazolidinediones reduce the LDL binding affinity of non-human primate vascular cell proteoglycans.

AIMS/HYPOTHESIS: Retention of atherogenic lipoproteins in the artery wall by proteoglycans is a key step in the development of atherosclerosis. Thiazolidinediones have been shown to reduce atherosclerosis in mouse models. The aim of this study was to determine whether thiazolidinediones modify vascular proteoglycan synthesis in a way that decreases LDL binding. METHODS: Primate aortic smooth muscle cells were exposed to troglitazone or rosiglitazone, or no stimulus at all for a 24-hour steady-state labelling period. Sulphate incorporation, size and LDL binding affinity of proteoglycans were determined. Proteoglycans secreted by cells in the presence or absence of troglitazone were separated into large and small classes by size exclusion chromatography, and LDL binding affinity was determined. RESULTS: Proteoglycans synthesised by cells exposed to troglitazone or rosiglitazone were smaller, with decreased sulphate incorporation and decreased LDL binding affinity. However, troglitazone had a greater effect than rosiglitazone. Troglitazone reduced the LDL binding affinities of both the large and small proteoglycans compared with control. The binding differences persisted when glycosaminoglycan chains released from proteoglycans were incubated with LDL, indicating that troglitazone affects the glycosaminoglycan synthetic machinery of these cells. CONCLUSIONS/INTERPRETATION: Thiazolidinediones decrease the LDL binding affinity of the proteoglycans synthesised by primate aortic smooth muscle cells. This could, in part, account for the reduced atherosclerosis observed in animal models.

Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor.

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF.

Cytochrome complex essential for photosynthetic oxidation of both thiosulfate and sulfide in Rhodovulum sulfidophilum.

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome c has been purified from the photosynthetic alpha-proteobacterium Rhodovulum sulfidophilum. This cytochrome c(551) is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome c(551) structural genes are part of an 11-gene sox locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome c(551) is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type R. sulfidophilum exhibit thiosulfate:cytochrome c oxidoreductase activity. However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c(551). Gene clusters similar to the R. sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

Novel heme ligation in a c-type cytochrome involved in thiosulfate oxidation: EPR and MCD of SoxAX from Rhodovulum sulfidophilum.

The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation. The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies. Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state. Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra. Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion. Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength. Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand. Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA. SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.

Troglitazone, but not rosiglitazone, inhibits Na/H exchange activity and proliferation of macrovascular endothelial cells.

Diabetes is associated with a high level of mortality due to cardiovascular disease resulting from accelerated coronary artery atherosclerosis. A current focus for investigation of atherosclerotic mechanisms is the vascular endothelium since physical or functional injury may represent an initiating step for atherogenesis. Thiazolidinediones (TZDs) are the newest class of drugs for the treatment of insulin resistance and its metabolic consequences; they are peroxisome proliferator-activating receptor (PPAR)-gamma ligands that act as insulin-sensitizing agents. We are interested in the contribution of direct vascular actions to the clinical utility of these agents. We investigated the effect troglitazone and rosiglitazone on endothelial cell proliferation in low- and high-glucose media and further explored their action on the ubiquitous membrane transport system, the Na/H exchanger (NHE), which has been implicated in regulating the growth of vascular cells. Experiments were conducted in cultured bovine aortic endothelial cells (BAECs). Cell proliferation was assessed by cell counting, and NHE activity was determined in cells loaded with the pH-sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). Troglitazone caused a dose-dependent inhibition of endothelial cell proliferation with approximately 50% inhibition at 10 microM. Troglitazone inhibited endothelial cell proliferation with similar potency under low- (5 mM) and high-glucose (25 mM) concentrations. Rosiglitazone had no significant effect on endothelial cell proliferation at concentrations of up to 100 microM under low- or high-glucose concentrations. The NHE inhibitor, 3-metlylsulfonyl-4-piperidinobenzoyl guanidine (HOE 694), caused dose dependent inhibition of BAEC proliferation, which was independent of the media glucose concentration. Acute exposure of cells to troglitazone (10 microM) and rosiglitazone (30 microM) during recovery from acidosis showed slight but significant (P<.05) inhibition of NHE activity by troglitazone, but no significant (P>.05) effect by rosiglitazone. Exposure of cells to either drug for 24 h revealed no chronic regulation of NHE activity. Our data demonstrate that troglitazone has similar actions in endothelial cells as in vascular smooth muscle. The absence of rosiglitazone effects, a more potent PPAR-gamma activator, suggests that the observed actions of troglitazone may be at least partially independent of PPAR-gamma. The effects of troglitazone and rosiglitazone on endothelial cell proliferation and NHE activity, although contrasting, are consistent with a central signalling role of this transporter in cell proliferation.

Diabetes-induced vascular hypertrophy is accompanied by activation of Na(+)-H(+) exchange and prevented by Na(+)-H(+) exchange inhibition.

Vascular disease often involves vessel hypertrophy with underlying cellular hypertrophy or hyperplasia. Experimental diabetes stimulates hypertrophy of the rat mesenteric vasculature, and we investigated the hypothesis that this hypertrophy is associated with activation of Na(+)-H(+) exchange (NHE) activity. We measured the NHE activity in isolated, intact blood vessels from control and streptozotocin-induced diabetic adult rats using concurrent myography and fluorescence spectroscopy. The role of inhibiting NHE activity in preventing the development of the mesenteric hypertrophy in streptozotocin-diabetic rats was investigated by administration of cariporide (100 mg/kg body weight per day in 3 doses by gavage) after induction of diabetes and subsequently determining vessel weight and structure. The weight of the mesenteric vasculature was not increased 1 week after streptozotocin treatment but was significantly increased by an average of 56% at 3 weeks. NHE activity in mesenteric arteries showed an enhanced maximal velocity (V:(max)) in diabetic vessels at 1 and 3 weeks (0.246+/-0.006 and 0. 238+/-0.007 versus 0.198+/-0.007 pH U/min) with no change in the apparent K:(m). Moreover, NHE-1 mRNA in mesenteric arterioles at 3 weeks after streptozotocin treatment was increased by >60% (55.8+/-6. 4 versus 91.3+/-12.3 fg). Administration of cariporide significantly reduced mesenteric vascular weight, the wall/lumen ratio, and mesenteric extracellular matrix accumulation in the diabetic animals. Our study shows that diabetes in vivo correlates with elevated NHE activity and mRNA in the mesenteric vasculature and furthermore that inhibition of this system prevents the hypertrophic response. These data suggest that NHE may be a target for therapeutic modulation of vascular changes in diabetes.

Effect of moderate alcohol upon obstructive sleep apnoea.

Moderate-to-large quantities of alcohol are known to aggravate severe obstructive sleep apnoea (OSA), however, the reported effects of moderate alcohol consumption upon mild-to-moderate OSA are inconsistent. Given the reported benefits of moderate alcohol consumption on cardiovascular mortality, recommendations regarding the management of patients with OSA are difficult to formulate. The aim of this study was to evaluate the effects of moderate alcohol on sleep and breathing in subjects with mild-to-moderate OSA. Twenty-one male volunteers, who snored habitually, underwent polysomnography with and without 0.5 g alcohol x kg body weight (BW)(-1) consumed 90 min prior to sleep time, in random order. The mean blood alcohol concentration (BAC) following alcohol at the time of lights out was 0.07 g x dL(-1). The distribution amongst the various sleep stages was not significantly altered by alcohol. The mean apnoea/hypopnoea index rose from 7.1+/-1.9 to 9.7+/-2.1 events x h(-1) (mean+/-SEM, p=0.017); however, there was no significant change in the minimum arterial oxygen saturation measured by pulse oximetry Sp,O2, apnoea length or snoring intensity. Mean sleep cardiac frequency rose significantly from 53.9+/-1.4 to 59.9+/-1.9 beats x min(-1) (P<0.001) and overnight urinary noradrenalin increased from 14.9+/-2.3 to 18.8+/-2.3 nmol x mmol creatinine(-1) (p=0.061) on the alcohol night compared to the nonalcohol night. To conclude, modest alcohol consumption, giving a mean blood alcohol concentration of 0.07 g x dL(-1), significantly increases both obstructive sleep apnoea frequency and mean sleep cardiac frequency.

Effects of sex and gonadectomy on cocaine metabolism in the rat.

The purpose of the current study is to determine whether sex differences in metabolism of cocaine (COC) exist that could contribute to the greater behavioral sensitivity of females to COC administration. To investigate this question, concentrations of COC and its two principle metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME) were measured by gas chromatography/mass spectroscopy in brain and plasma collected from male and female rats that were sacrificed between 5 and 90 min after injection COC (15 mg/kg i.p.). COC concentrations did not differ in plasma or brain tissue of males and females, but sex-specific patterns of metabolite distribution were detected. BE was 2-fold higher in plasma and brain of males than females, whereas EME was much higher in brain and plasma of females. The influence of gonadal hormones on COC metabolite patterns were determined using gonadectomized and prepubertal rats. Castration of male or female rats did not alter brain or plasma COC, but did decrease BE concentrations. Seven-day-old pups injected with 15 mg/kg of COC had higher blood and brain COC than adults and relatively low levels of metabolites. No sex differences were found for COC, BE, or EME in brain or plasma of pups. These findings indicate that although gonadal steroids influence COC metabolism, these effects do not explain sex differences in COC-induced behaviors.

Rapid regulation of adrenomedullin in metabolically compromised vascular smooth muscle cells.

OBJECTIVE: The prepro-adrenomedullin gene encodes the biologically active peptide adrenomedullin, which acts as a potent vasodilator as well as a modulator of vascular smooth muscle cell growth. We investigated the question of whether adrenomedullin is regulated in response to metabolic perturbations in vascular smooth muscle. MATERIALS AND METHODS: Acute inhibition of glycolysis, leading to partial depletion of cellular ATP, was produced in cultured rat aortic vascular smooth muscle cells by replacing glucose with 2-deoxyglucose. Solution hybridization/RNase protection analysis was used to quantitate changes in expression of the prepro-adreno-medullin messenger RNA and a specific radioimmunoassay was used to assess levels of secreted adrenomedullin. RESULTS: Acute incubation of rat aortic vascular smooth muscle cells with 2-deoxyglucose caused a rapid and sustained induction of low basal levels of adrenomedullin messenger RNA, which reached twice the control levels by 1 h and four times control levels by 6 h. The induction of adrenomedullin messenger RNA expression was dependent upon de-novo gene transcription and was reversed by the re-introduction of glucose. Despite the sustained increase in adrenomedullin messenger RNA, secretion of immunoreactive-adrenomedullin from vascular smooth muscle cells was reduced by as much as 75% and paralleled the inhibition of radiolabeled amino acid incorporation into protein during glycolytic inhibition; both parameters recovered towards control levels following re-introduction of glucose. CONCLUSIONS: The rapid and reversible activation of the adrenomedullin gene and inhibition of adrenomedullin peptide release in response to metabolic inhibition suggest that adrenomedullin represents a novel localized mechanism that may modulate regional blood flow and vascular smooth muscle cell proliferation in response to perturbations of normal metabolism.

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responses following endothelin-1 stimulation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in [3H]Ins(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense- or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1, 4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4, 5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.

Identification of a novel, inhibitory action of amiodarone on vesicular monoamine transport.

The benzofuran antiarrhythmic drug, amiodarone, exhibits a wide range of pharmacological properties. Recent in vivo biochemical studies suggest that amiodarone may exert an antiadrenergic action in the heart, which resembles the effects of reserpine. To investigate the cellular basis for this apparent presynaptic, sympatholytic action we used Chinese hamster ovary (CHO) cells expressing the type 2 vesicular monoamine transporter (VMAT2) as a synaptic vesicular model. Amiodarone inhibited the uptake of [3H]norepinephrine in VMAT2-transfected CHO cells in a concentration-dependent manner, with a -log EC50 of 6.44 +/- 0.32. To further identify the site at which amiodarone suppressed vesicular monoamine transport, we examined the ability of amiodarone to displace [3H]reserpine from its binding site in membrane fractions prepared from CHO cells expressing VMAT2. [3H]Reserpine binding was inhibited in a concentration-dependent manner by amiodarone, with an -log EC50 of 6.76 +/- 0.03, reaching 84 +/- 5% inhibition of reserpine binding at 10 microM. A pH-dependent mechanism for this action of amiodarone was excluded in studies using the pH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF). These data indicate that amiodarone inhibits the uptake of monoamine into the axoplasmic storage vesicle by inhibiting VMAT. Furthermore, amiodarone competes specifically with reserpine for binding to VMAT. These findings suggest a novel presynaptic site of action for amiodarone.

Protection of neuronal uptake-1 inhibitors in ischemic and anoxic hearts by norepinephrine-dependent and -independent mechanisms.

Cardiac ischemia and anoxia induce massive norepinephrine (NE) release, which is mediated by a reverse operation of uptake-1 and can be suppressed by uptake-1 inhibitors. We studied effects of uptake-1 inhibitors on incidence of ventricular fibrillation (VF%) and myocardial contracture in perfused rat hearts under ischemic or anoxic conditions. NE release occurred in hearts during ischemia or anoxia and was largely inhibited by desipramine, imipramine, and cocaine. The generation of inositol 1,4,5-trisphosphate (InsP3) during reperfusion also was abolished by desipramine. During anoxia/reoxygenation, VF (93 and 71%, respectively) and myocardial contracture occurred and were significantly inhibited by desipramine and by NE depletion. Regional ischemia and reperfusion induced high VF% (86 and 100%, respectively), which was reduced or abolished by desipramine and imipramine at 0.03 and 0.3 microM. During the ischemic phase, cocaine was similarly antiarrhythmic, as was a combination of timolol and prazosin, but NE depletion was not. In NE-depleted hearts, cocaine or the combination of timolol and prazosin showed limited effect on VF%, whereas both desipramine and imipramine abolished VF. In anesthetized rats in vivo, ischemic VF% was reduced by desipramine (30 vs. 92%; p < 0.01). In conclusion, uptake-1 inhibitors protect hearts against ischemia/reperfusion- and anoxia/reoxygenation-induced arrhythmias, partly because of the inhibition of locally mediated NE release. Other actions of desipramine and imipramine may contribute to the overall efficacy.

Developmental differences in the acquisition of tolerance to ethanol.

This study was designed to compare the development of tolerance to ethanol in adolescent and adult rats. Rats were pretreated with ethanol (4 g/kg) twice daily by intragastric gavage for 3 or 7 days, and then challenged with a single IP dose of ethanol (5 g/kg). Throughout the pretreatment period body temperature was measured before and after the morning dosage. During the IP challenge test we measured body temperature, duration of the loss of righting reflex, and blood ethanol level upon regaining the righting reflex. The adolescent rats that were pretreated for 7 days developed greater tolerance to the effects of ethanol on body temperature during the pretreatment period. No tolerance was observed in animals that received only 3 days of pretreatment. Ethanol decreased body temperature to a greater extent in adolescent animals than adults in response to the IP challenge dose, but there was no significant difference between control and pretreated animals in either age group. The time to regain the righting reflex was lower in adolescent rats than in adults. Post hoc analyses indicated that 7 days of ethanol pretreatment diminished the effects of the IP ethanol challenge on the loss of righting reflex in adolescent but not adult rats. Although adolescent rats regained the righting reflex with higher blood ethanol levels than adults, there was no significant effect of ethanol pretreatment on that measure. These findings indicate that the development of tolerance to the temperature regulatory effects of ethanol is more marked in the adolescent rat than in the adult. This developmental difference in ethanol sensitivity is consistent with other recent findings and suggests that adolescence may be a period of unique sensitivity to a number of the behavioral and physiological effects of ethanol.

Mechanisms regulating the vascular smooth muscle Na/H exchanger (NHE-1) in diabetes.

Vascular disease is a major component of the complications associated with diabetes. The pathology involves hypertrophy and proliferation of vascular smooth muscle cells and the production and modification of extracellular matrix. The sodium/hydrogen exchanger has been widely implicated in the growth of multiple cell types, including vascular smooth muscle. Increases in sodium/hydrogen exchange activity serve as an effector or at least as an indicator of vascular activation. This article is concerned with the role of the biochemical abnormalities of diabetes exerting their pathological effects on vascular smooth muscle cells via altering sodium/hydrogen exchange activity.

Urokinase plasminogen activator induces smooth muscle cell migration: key role of growth factor-like domain.

We defined the role of urokinase plasminogen activator (uPA) and its growth factor-like domain (GFD) in stimulating smooth muscle cell (SMC) migration. Recombinant uPA (r-uPA) stimulated migration approximately 3-fold whilst the recombinant uPA mutant containing an altered GFD (r-uPAmut) was ineffective. Both uPA variants bound to the same high affinity receptor in a competitive manner. FGF-2- and PDGF-BB-induced migration was also dependent on uPA, their effects being antagonized by addition of a uPA-neutralizing antibody or the r-uPAmut. Thus r-uPA is chemotactic for SMC and stimulation of cell migration by PDGF-BB and FGF-2 is dependent on uPA. The GFD of uPA is essential for its chemotactic effects.