Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing.

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol®) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

Inhibition of platelet aggregation ex vivo is repressed in apolipoprotein E deficient mice.

In the present study, we assessed whether the endogenous platelet inhibitory mechanisms are altered in the early to moderate stages of the atherosclerotic process. Apolipoprotein E deficient mice (ApoE-/-), a mouse model of atherosclerosis, and their wild-type (WT) counterparts were used to assess agonist-stimulated synthesis of prostacyclin (PGI2), inhibition of platelet aggregation ex vivo, and intra-platelet cAMP levels. Basal U46619 and ADP -induced platelet aggregation in vitro were increased in ApoE-/- mice at 18-20 weeks in comparison with 8-10 weeks of age. Systemically administered endothelin-1 (ET-1) or bradykinin (BK) inhibited platelet aggregation in a similar fashion in 8- to 10-week-old ApoE-/- and WT mice, but not in the ApoE-/- mice at 18-20 weeks of age, although both peptides maintained their capacity to increase plasma levels of the PGI2. Intravenous infusion of PGI2 also failed to inhibit platelet aggregation ex vivo in 18- to 20-week-old ApoE-/- mice. Interestingly, both BK and PGI2 retained their ability to increase intraplatelet cAMP in WT and ApoE-/- mice. Our results suggest that a loss of activity of endogenous inhibitorymechanisms could contribute to the increased platelet reactivity in ApoE-/- mice, and that this phenomenon occurs early in the intermediate stage of the atherosclerotic process.

Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides.

L-arginine causes insulin release from pancreatic B cells. Data from three model systems support the hypothesis that L-arginine-derived nitrogen oxides (NOs) mediate insulin release stimulated by L-arginine in the presence of D-glucose and by the hypoglycemic drug tolbutamide. The formation of NO in pancreatic B cells was detected both chemically and by the NO-induced accumulation of guanosine 3',5'-monophosphate. NG-substituted L-arginine analogs inhibited the release of both insulin and NO. Protein immunoblot and histochemical analysis with antiserum to type I NO synthase suggest that the formation of NO in pancreatic B cells is catalyzed by an NADPH- (reduced form of nicotinamide adenine dinucleotide phosphate), Ca2+/calmodulin-dependent type I NO synthase of about 150 kilodaltons.

Platelet responses to pharmacological and physiological interventions in middle-aged men with different habitual physical activity levels.

The current guidelines following an acute coronary syndrome recommend dual-antiplatelet therapy (DAPT) (aspirin plus a P2Y12 antagonist) alongside lifestyle modifications, including more regular physical activity. It is currently unknown whether regular exercise affects the pharmacology of DAPT. AIM: To explore how exercise-induced improvements in vascular and platelet function affect the efficacy of DAPT, in a cross-sectional study of men with different physical activity levels (training status). METHODS: A total of 42 healthy, normal-weight, middle-aged men were divided into 3 groups: untrained, moderately trained and well-trained. Their platelet reactivity (agonist-induced % aggregation) was investigated in platelet-rich plasma at rest and after inhibition with aspirin and ticagrelor and/or prostacyclin and nitric oxide added to the blood in vitro, and after physiological tests of vascular function; passive movement of the leg, flow-mediated dilation and one-leg knee-extensor exercise. Vascular function of the femoral artery (changes in arterial blood flow) was assessed by ultrasound Doppler. RESULTS: Platelets from the well-trained subjects had lower basal reactivity, a higher sensitivity to the anti-aggregatory effects of prostacyclin and were more potently inhibited by DAPT compared to the untrained subjects. The moderately trained and well-trained subjects had a superior vascular function compared to untrained subjects, and their platelets were more inhibited by the passive movement, flow-mediated dilation and one-leg knee-extensor exercise. DISCUSSION: A habitually active lifestyle leads to an increased platelet sensitivity to pharmacological and physiological platelet inhibitors. We suggest that physical activity habits (training status) should be considered when personalizing and optimizing antithrombotic treatment strategies.

Why Neighborhoods (and How We Study Them) Matter for Adolescent Development.

Adolescence is a sensitive developmental period marked by significant changes that unfold across multiple contexts. As a central context of development, neighborhoods capture-in both physical and social space-the stratification of life chances and differential distribution of resources and risks. For some youth, neighborhoods are springboards to opportunities; for others, they are snares that constrain progress and limit the ability to avoid risks. Despite abundant research on "neighborhood effects," scant attention has been paid to how neighborhoods are a product of social stratification forces that operate simultaneously to affect human development. Neighborhoods in the United States are the manifestation of three intersecting social structural cleavages: race/ethnicity, socioeconomic class, and geography. Many opportunities are allocated or denied along these three cleavages. To capture these joint processes, we advocate a "neighborhood-centered" approach to study the effects of neighborhoods on adolescent development. Using nationally representative data from the National Longitudinal Study of Adolescent to Adult Health (Add Health), we demonstrate the complex ways that these three cleavages shape specific neighborhood contexts and can result in stark differences in well-being. A neighborhood-centered approach demands more rigorous and sensitive theories of place, as well as multidimensional classification and measures. We discuss an agenda to advance the state of theories and research, drawing explicit attention to the stratifying forces that bring about distinct neighborhood types that shape developmental trajectories during adolescence and beyond.

California dreamin' 'bout endothelin: emerging new therapeutics.

Progress in our understanding of endothelins has been extremely rapid since their discovery in 1988. A number of pharmaceutical companies have developed potent, orally active, nonpeptide endothelin receptor antagonists with efficacies in a wide variety of animal disease models and potential for treating human disease. However, only time will tell whether or not these compounds are developed into drugs that are perceived as worthy of a place in the therapeutic marketplace. If this is the case, endothelin will have been promoted from the status of striking scientific discovery to therapeutic target in a remarkably short period.

Internal medicine, psychiatry, and emergency medicine residents' views of assisted death practices.

BACKGROUND: Although studies have revealed conflicting attitudes within the medical community regarding assisted death practices in the United States, the views of current resident physicians have not been described. OBJECTIVE: To investigate the perspectives of residents from 3 medical specialty fields regarding the acceptability of assisted suicide and euthanasia practices as performed by 4 possible agents (the resident personally, a referral physician, physicians in general, or nonphysicians in general) in 6 patient scenarios. METHODS: An anonymous survey exploring responses to 6 patient vignettes was conducted with a convenience sample of all residents in the internal medicine, psychiatry, and emergency medicine training programs. RESULTS: A total of 96 residents, 72% of those asked, participated in this study. Overall, residents expressed opposition or uncertainty regarding assisted suicide and euthanasia. The residents were disinclined to directly perform such practices themselves and did not support the conduct of assisted suicide practices by nonphysicians. Respondents were somewhat more accepting of other physicians' involvement in assisted death activities. Conflicting views were expressed by residents, with emergency medicine residents more likely to support assisted suicide practices in 4 of 6 patient vignettes than either internal medicine or psychiatry residents. Residents who reported being influenced by religious beliefs (21 respondents [22%]) did not support assisted death practices, whereas those influenced by personal philosophy (74 respondents [77%]) expressed less opposition. CONCLUSIONS: This study explores the uncertainty and differing views of residents from 3 fields about physician-assisted suicide practices. Study findings are considered within the larger literature on clinician attitudes toward assisted suicide and euthanasia.

Complex insomnia: insomnia and sleep-disordered breathing in a consecutive series of crime victims with nightmares and PTSD.

BACKGROUND: Sleep disturbance in posttraumatic stress disorder is very common. However, no previous posttraumatic stress disorder studies systematically examined sleep breathing disturbances, which might influence nightmares, insomnia, and posttraumatic stress disorder symptoms. METHODS: Forty-four consecutive crime victims with nightmares and insomnia underwent standard polysomnography coupled with a nasal pressure transducer to measure airflow limitation diagnostic of obstructive sleep apnea and upper airway resistance syndrome. RESULTS: Forty of 44 participants tested positive on objective sleep studies based on conservative respiratory disturbance indices of more than 15 events per hour; 22 patients suffered from obstructive sleep apnea and 18 suffered from upper airway resistance syndrome. CONCLUSIONS: In an uncontrolled study, insomnia and sleep-disordered breathing were extremely prevalent in this small and select sample of crime victims. Research is needed to study 1) prevalence of sleep-disordered breathing in other posttraumatic stress disorder populations using appropriate controls and nasal pressure transducers and 2) effects of sleep treatment on posttraumatic stress symptoms in trauma survivors with comorbid obstructive sleep apnea or upper airway resistance syndrome. In the interim, some posttraumatic stress disorder patients may benefit from sleep medicine evaluations.

Perspectives of patients with schizophrenia and psychiatrists regarding ethically important aspects of research participation.

OBJECTIVE: Significant controversy surrounds the ethics of psychiatric research. Nevertheless, few data have been gathered to improve our understanding of how individuals with serious mental illness and psychiatrists view ethically important aspects of biomedical research participation. METHOD: The authors assessed views of clinically diagnosed patients with schizophrenia from three sites by means of structured interviews and views of psychiatrists at two sites with written surveys regarding attitudes affecting motivation to participate in biomedical research, attitudes related to autonomy and influences on participation decisions, and attitudes toward the inclusion of vulnerable populations in research. The schizophrenia patients were asked to indicate their personal views; the psychiatrists were asked to provide their personal views and to predict schizophrenia patients' views. Responses were compared by using repeated measures multivariate analysis of variance. RESULTS: Sixty-three patients with schizophrenia and 73 psychiatry faculty and residents participated. Overall, responses to 23 rated attitudes revealed remarkably similar rank orders and several areas of agreement between patients and psychiatrists. Both groups strongly supported schizophrenia research and autonomous decision making by participants. They saw helping others and helping science as important reasons for protocol participation. Patients endorsed the feeling of hope associated with research involvement, a perspective underestimated by psychiatrists. Psychiatrists also underestimated the patients' acceptance of physician, investigator, and family influences on participation decisions. Psychiatrists agreed more strongly than patients that vulnerable populations should be included in research. CONCLUSIONS: This study helps to characterize previously neglected attitudes of psychiatric patients and clinicians toward ethically important aspects of biomedical research participation. Schizophrenia patients offered highly discerning views, and interesting similarities and differences emerged in comparing responses of patients and psychiatrists.

An open-label trial of evidence-based cognitive behavior therapy for nightmares and insomnia in crime victims with PTSD.

OBJECTIVE: Insomnia and nightmares are perceived as secondary phenomena in posttraumatic stress disorder (PTSD). Scant treatment research has targeted these two sleep disturbances. This study reports on an open-label trial of cognitive behavior therapy for insomnia and disturbing dreams in crime victims with PTSD. The relationship among nightmares, sleep disturbances, and PTSD symptoms is discussed. METHOD: Sixty-two participants completed a 10-hour group treatment consisting of imagery rehearsal for nightmares and sleep hygiene, stimulus control, and sleep restriction for insomnia. Nightmare frequency, sleep quality, sleep impairment, and ratings for PTSD, anxiety, and depression symptoms were assessed at baseline and at the 3-month follow-up. RESULTS: All measures demonstrated improvement that was roughly equivalent to changes in clinical severity from severe to moderate for sleep quality, sleep impairment, and nightmare frequency, from borderline severe to borderline moderate for PTSD symptoms, and from extremely severe to borderline severe for anxiety and depression symptoms. CONCLUSIONS: In this uncontrolled study, successful treatment for insomnia and nightmares in crime victims was associated with improvement in symptoms of PTSD, anxiety, and depression. Participants with clinical improvements in PTSD symptoms demonstrated significantly greater improvement in sleep quality and nightmare frequency than those whose PTSD symptoms did not improve.

Simultaneous perfusion of rat isolated superior mesenteric arterial and venous beds: comparison of their vasoconstrictor and vasodilator responses to agonists.

1. A new isolated perfused preparation is described that allows a direct comparison to be made of the responses of the perfused arterial and retrogradely perfused venous circulations of the rat superior mesenteric vascular bed. 2. In experiments comparing the responses of the intact arterially perfused mesentery and small intestine to those of the same preparation following removal of the intestine and division of the circulations, the increases in perfusion pressure produced by arginine-vasopressin (30 pmol) and noradrenaline (1 nmol) were retained by the arterial circulation and those induced by angiotensin II (30 pmol) by the venous circulation. Endothelin-1 (30 pmol) constricted both portions of the vasculature but the prolonged nature of its response was associated with only the venous vessels. 3. In the simultaneously perfused arterial and venous preparation arginine vasopressin (3-100 pmol) was a selective constrictor of the arterial circulation and angiotensin II (3-100 pmol) of the venous circulation. In addition, noradrenaline (0.3-10 nmol), 5-hydroxytryptamine (0.3-10 nmol) and KCl (1-60 micromol) were more active as constrictors of the arterial than the venous vessels, and U46619 (10-300 pmol) a more active constrictor of the venous than the arterial vessels. Endothelin-1 (3-100 pmol) constricted both the arterial and venous portions of the vasculature but was significantly longer acting as a venoconstrictor than an arterioconstrictor. 4. Angiotensin I (300 pmol) caused constrictions of the venous circulation which were dependent upon the presence of angiotensin converting enzyme for captopril (10 microM) abolished constrictions caused by angiotensin I but not by angiotensin II. 5. In preparations preconstricted by U46619 (0.3-3 microM), acetylcholine (0.01-100 nmol), bradykinin (0.001-nmol), sodium nitroprusside (0.01-lOnmol) or isoprenaline (1-l00pmol) produced dose-related dilatations of both the arterial and the venous vasculatures, whereas adenosine diphosphate (ADP, 0.01-lOOnmol) caused dose-dependent dilatations of the arterial circulation but principally constrictions of the venous circulation. The dilatations caused by acetylcholine and bradykinin in both portions of the circulation, and by ADP in the arterial circulation, were endothelium-dependent as they were inhibited by gossypol (3 microM), whereas dilatations to sodium nitroprusside were not. 6. This preparation allows the responses of the arteries and veins of a single perfused mesenteric bed to be compared. In addition, with this preparation it is possible to demonstrate that veins, as well as arteries, show significant endothelium-dependent relaxations. It is concluded that the venous portion of the vasculature is significantly involved in the responses of the intact circulation.

Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-inflammatory drugs.

1. In this study we describe experiments to establish ex vivo the selectivity of non-steroidal anti-inflammatory drugs (NSAIDs) given in vivo. 2. Anaesthetised (Inactin, 120 mg kg(-1)) male Wistar rats (220-250 g) received an i.v. dose of one of the following compounds (dose mg kg(-1)): aspirin (20), diclofenac (3), L-745,337 (30), nimesulide (15), salicylate (20), sulindac (10). Blood samples were taken before and up to 6 h after dosing and the plasma obtained from it was tested for its ability to inhibit prostanoid formation in IL-1beta-treated A549 cells (COX-2 system) and human washed platelets (COX-1 system). For control the same compounds were also added directly to the assay systems. 3. All drugs, except sodium salicylate, inhibited COX-1 and COX-2 when added directly to the test systems. Plasma from aspirin-treated rats was without effect on either COX-1 or COX-2, consistent with the rapid in vivo metabolism to salicylate. Conversely, plasma from sulindac-treated rats inhibited COX-1 and COX-2 with potencies according with in vivo metabolism to sulindac sulphide. Diclofenac was COX-1/2 non-selective when tested in vitro, but a slightly preferential inhibitor of COX-2 when tested ex vivo. Nimesulide was confirmed as preferential inhibitor of COX-2 both in vitro and ex vivo. L-745,337 was a selective COX-2 inhibitor when tested in vitro or ex vivo. 4. In conclusion, our experiments show clearly (a) NSAIDs inactivation, (b) activation of prodrugs, and (c) NSAIDs selectivity. Our assay provides useful information about the selectivity of NSAIDs that could be extended by the analysis of plasma samples taken from humans similarly treated with test drugs.

Interactions between inducible isoforms of nitric oxide synthase and cyclo-oxygenase in vivo: investigations using the selective inhibitors, 1400W and celecoxib.

1. Exposure of tissues to endotoxin (LPS) and/or cytokines leads to the induction of both inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). It has previously been reported that there is 'cross-talk' between these two systems. However, such previous studies have been limited by the availability of highly selective inhibitors. Here we have investigated the interactions between iNOS and COX-2 in vivo using 1400W, an iNOS-selective inhibitor, and celecoxib, a COX-2-selective inhibitor. 2. Infusion of LPS to rats for 6 h caused a time-dependent increase in the plasma concentrations of 6 keto-prostaglandin F1alpha (6 keto-PGF1alpha) and nitrite/nitrate (NO2/NO3), consistent with the induction of iNOS and COX-2. Bolus injection of arachidonic acid (AA) at t=6 h resulted in a further increase of circulating levels of 6 keto-PGF1alpha in LPS-treated animals. 3. Treatment of rats with 1400W or the non-selective NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibited the increase in plasma NO2/NO3 but were both without effect on the plasma concentration of 6 keto-PGF1alpha before or after AA. 4. Treatment with the non-steroidal anti-inflammatory drugs (NSAIDs), A771726 or diclofenac, or with celecoxib significantly reduced the increase in circulating 6 keto-PGF1alpha caused by LPS, and the large increase in 6 keto-PGF1alpha following injection of AA. None of the COX inhibitors affected the increase in plasma NO2/NO3. Dexamethasone, however, significantly inhibited both the increase in 6 keto-PGF1alpha and the increase in NO2/NO3. 5. In conclusion, the use of selective inhibitors does not support the concept of cross talk in vivo between iNOS and COX-2.

Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy.

Cyclo-oxygenase is expressed in cells in two distinct isoforms. Cyclo-oxygenase-1 is present constitutively whilst cyclo-oxygenase-2 is expressed primarily after inflammatory insult. The activity of cyclo-oxygenase-1 and -2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo-oxygenase include the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo-oxygenase-2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo-oxygenase-1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo-oxygenase-2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo-oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Involvement of endothelin in the pressor response following injection of NMDA to the periaqueductal gray area of rats.

Microinjection of N-methyl-D-aspartate (NMDA) (0.068 to 6.8 nmol) into the periaqueductal gray area (PAG) of anaesthetized rats caused dose-dependent increases in blood pressure. Preinjection (10 min before) of FR 139317 (an ETA receptor selective antagonist; 5 nmol) or SB 209670 (an ETA/ETB receptor non-selective antagonist; 5 nmol) to the PAG reduced the pressor response to NMDA whereas BQ-788 (an ETB receptor selective antagonist; 5 nmol) did not affect the NMDA-induced hypertension. Pretreatment with DL-2-amino-5-phosphono valeric acid (2-APV) (an NMDA receptor selective antagonist, 5 nmol) also abolished the pressor response induced by NMDA. Dose-dependent increases in blood pressure induced by injection of angiotensin II (0.1-10 nmol) to the PAG were unaffected by FR 139317 or SB 209670. Thus, our data indicate that endogenous ET-1, via an action on ETA receptors, contributes to the pressor effects of NMDA within the brain.

A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo-oxygenase-2 in vitro and in vivo in a substrate-sensitive manner.

1. The immunosuppressive and anti-inflammatory drug leflunomide has several sites of action, although its precise mode of action is unknown. 2. Here we show in vitro and in vivo that leflunomide and/or its active metabolite A771726, inhibit the activity of cyclo-oxygenase (COX) at doses below those that affect protein expression. 3. In J774.2 macrophages treated with endotoxin for 24 h to induce COX-2 and iNOS, leflunomide and A771726 inhibited more potently the accumulation of PGE2 (A771726, IC50 3.5 microg ml-1) than of NO2 (A771726, IC50 380 microg ml-1). At high concentrations (>300 microg ml-1) A771726 also exhibited the expression of COX-2 and iNOS proteins. 4. In A549 cells treated for 24 h with interleukin-1beta, to induce COX-2, A771726 potently inhibited PGE2 synthesis (IC50 0.13 microg ml-1). In the same cells, A771726 was notably less active (IC50, 52 microg ml-1) at inhibiting the formation of PGE2 stimulated by exposure to 30 microM arachidonic acid. 5. In a human whole blood assay, measuring the accumulation of TxB2 in response to calcium ionophore as a measure of COX-1 activity and in response to incubation with bacterial endotoxin as a measure of COX-2 activity, leflunomide inhibited COX-1 and COX-2 with IC50 values of 31 and 185 microg ml-1; for A771726 the corresponding values were 40 and 69 microg ml-1. 6. Pre-treatment of rats with leflunomide or A771726 (10 mg kg-1, i.p.) inhibited the plasma accumulation of 6-keto-PGF1alpha but not NO2/NO3 following infusion of endotoxin. Injection of a bolus of arachidonic acid following 6 h infusion of endotoxin caused a marked acute rise in plasma 6-keto-PGF1alpha which was inhibited only by higher doses of A771726 (50 mg kg-1, i.p.). 7. In conclusion, leflunomide via A771726 can directly inhibit the activity of COX, an effect that appears blunted both by increases in substrate supply and possibly by plasma binding. Only at much higher drug levels does leflunomide and/or A771726 inhibit the induction of COX-2 or iNOS proteins.

Bisphenol A diglycidyl ether (BADGE) is a PPARgamma agonist in an ECV304 cell line.

Peroxisome proliferator activated receptors (PPAR)s are nuclear transcription factors of the steroid receptor super-family. One member, PPARgamma, a critical transcription factor in adipogenesis, is expressed in ECV304 cells, and when activated participates in the induction of cell death by apoptosis. Here we describe a clone of ECV304 cells, ECV-ACO.Luc, which stably expresses a reporter gene for PPAR activation. ECV-ACO.Luc respond to the PPARgamma agonists, 15-deoxy-Delta(12,14) PGJ(2), and ciglitizone, by inducing luciferase expression. Furthermore, using ECV-ACO.Luc, we demonstrate that a newly described PPARgamma antagonist, bisphenol A diglycidyl ether (BADGE) has agonist activities. Similar to 15-deoxy-Delta(12,14) PGJ(2), BADGE induces PPARgamma activation, nuclear localization of the receptor, and induces cell death.

Comparison of the survival of endothelium-derived relaxing factor and nitric oxide within the isolated perfused mesenteric arterial bed of the rat.

1. The survival of the endothelium-derived relaxing factor (EDRF) released by acetylcholine (ACh) in the rat isolated perfused mesenteric vascular bed was compared to that of exogenously applied nitric oxide (NO) by direct bioassay of the effluent from the mesenteric bed on rabbit aortic strips (RbAs). 2. NO (0.1-10 nmol) produced dose-related vasodilatations in the mesenteric vascular bed and also survived in the effluent in concentrations sufficient to provoke relaxations of the RbAs. ACh (1.7 pmol-300 nmol) caused dose-related vasodilatations in the mesenteric bed but no EDRF was detected in the effluent. This was true even when ACh provoked much larger vasodilatations in the mesenteric bed than those to doses of NO that survived passage through the bed. 3. Removal of the endothelial cells in the mesenteric vascular bed abolished vasodilatations in response to ACh but did not significantly affect those to NO. Survival of NO through the mesenteric bed was not increased. 4. Superoxide dismutase (SOD, 10 u ml-1) did not significantly affect the vasodilator effects of either ACh or NO. It did increase the survival of NO through the mesenteric vascular bed but native EDRF was still not detected in the effluent. 5. The absence of bioassayable EDRF in the effluent following ACh-induced vasodilatations might be explained by the volume into which EDRF is released abluminally being much smaller than that into which it is released luminally.

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

Endothelin-1 and the periaqueductal gray area of the rat: an autoradiographic and functional pharmacological study.

Endothelin-1 (ET-1) injected centrally induces pressor effects and associated haemodynamic changes. Here we have evaluated the effects on systemic and regional cardiovascular parameters of injection of ET-1 into the periaqueductal gray (PAG) area of anaesthetized rats. In addition, we have used the ETA receptor-selective antagonist, FR 139317, the ETB receptor-selective antagonist, BQ-788, and the ETA/ ETB receptor non-selective antagonist, SB 209670, to identify the receptor(s) mediating these effects. We have also used in vitro autoradiography to identify binding sites for ET-1 in the PAG. 2. In vitro autoradiography showed dense binding of [125I]-PD 151242 (for ETA receptors) in the PAG area, with the binding sites being homogeneously distributed within the dorsal, lateral and ventral subregions. Tissues incubated with [125I]-BQ 3020 (for ETB receptors) had little binding. 3. Injection of ET-1 (0.1, 1 and 10 pmol per rat) in the dorsolateral PAG area significantly increased, in a dose-dependent manner the mean arterial blood pressure (MAP). The highest dose of ET-1 (10 pmol) also decreased the heart rate by 18 +/- 1%, n = 6 (P < 0.05). Increases in blood pressure induced by ET-1 (1 pmol; 31 +/- 6.6 mmHg, n = 6) were greatly reduced by pre-administration to the PAG area of FR 139317 (5 nmol per rat) or SB 209670 (3 nmol per rat) (97 and 94%, respectively), but were unaffected by BQ-788 (5 nmol per rat). Similarly, FR 139317 and SB 209670 prevented the decrease in heart rate induced by ET-1 while BQ-788 did not affect it. 4. Injection of ET-1 to the PAG area caused falls in renal blood flow (RBF) as measured by an ultrasonic flow probe, and increased renal vascular resistance (RVR). Pre-treatment of the PAG with FR 139317 or SB 209670, but not with BQ-788, prevented this ET-1-induced effect. 5. Injection of ET-1 (10 pmol) also increased total peripheral resistance (TPR; control, 2.39 +/- 0.2 mmHg ml-1 min 100 g body weight) by 100 +/- 9% (n = 5) and reduced the cardiac output (CO; control, 94.7 +/- 3.1 ml min-1) by 30 +/- 3% (n = 5), as determined by radioactive microspheres. Vascular resistances were increased in other organs, such as skeletal muscle (88 +/- 5%, n = 4), the colon (55 +/- 7%, n = 4) and the stomach (47 +/- 3%, n = 4). Pretreatment of the PAG area with FR 139317 or SB 209670 reduced the increases in TPR and vascular resistance, and the reduction in CO caused by ET-1. BQ-788 did not effect the responses to ET-1. 6. Thus, there are predominantly ETA binding sites within the PAG area and injection of ET-1 into the PAG area causes complex haemodynamic changes which are sensitive to ETA receptor antagonism. ETA receptors are, therefore, the predominant mediators of the actions of ET-1 in the PAG of the rat.