P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.

Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction.

BACKGROUND: Numerous pathological mediators of cardiac hypertrophy (eg, neurohormones, cytokines, and stretch) have been shown to activate p38 MAPK. The purpose of the present study was to examine p38 MAPK activation and the effects of its long-term inhibition in a model of hypertensive cardiac hypertrophy/dysfunction and end-organ damage. METHODS AND RESULTS: In spontaneously hypertensive stroke-prone (SP) rats receiving a high-salt/high-fat diet (SFD), myocardial p38 MAPK was activated persistently during the development of cardiac hypertrophy and inactivated during decompensation. Long-term oral treatment of SFD-SP rats with a selective p38 MAPK inhibitor (SB239063) significantly enhanced survival over an 18-week period compared with the untreated group (100% versus 50%). Periodic echocardiographic analysis revealed a significant reduction in LV hypertrophy and dysfunction in the SB239063-treatment groups. Little or no difference in blood pressure was noted in the treatment or vehicle groups. Basal and stimulated (lipopolysaccharide) plasma tumor necrosis factor-alpha concentrations were reduced in the SB239063-treatment groups. In vitro vasoreactivity studies demonstrated a significant preservation of endothelium-dependent relaxation in animals treated with the p38 MAPK inhibitor without effects on contraction or NO-mediated vasorelaxation. Proteinuria and the incidence of stroke (53% versus 7%) were also reduced significantly in the SB239063-treated groups. CONCLUSIONS: These results demonstrate a crucial role for p38 MAPK in hypertensive cardiac hypertrophy and end-organ damage. Interrupting its function with a specific p38 MAPK inhibitor halts clinical deterioration.

Pharmacology of SB-273779, a nonpeptide calcitonin gene-related peptide 1 receptor antagonist.

Calcitonin gene-related peptide (CGRP), a potent vasodilatory and cardiotonic peptide, has a potential role for CGRP in diverse physiologic and pathophysiologic situations such as congestive heart failure, diabetes, migraine, and neurogenic inflammation. Although a peptide CGRP receptor antagonist, CGRP(8-37,) is available, its utility presents significant limitations for these indications. Here, we describe the properties of SB-(+)-273779 [N-methyl-N-(2-methylphenyl)-3-nitro-4-(2-thiazolylsulfinyl)nitrobenzanilide], a selective nonpeptide antagonist of CGRP(1) receptor. SB-(+)-273779 inhibited (125)I-labeled CGRP binding to SK-N-MC (human neuroblastoma cells) and human cloned CGRP(1) receptor with K(i) values of 310 +/- 40 and 250 +/-15 nM, respectively. SB-(+)-273779 also inhibited CGRP (3 nM)-activated adenylyl cyclase in these systems with IC(50) values of 390 +/-10 nM (in SK-N-MC) and 210 +/-16 nM (recombinant human CGRP receptors). Prolonged treatment (>30 min) of SK-N-MC cells with SB-(+)-273779 followed by extensive washing resulted in reduction in maximum CGRP-mediated adenylyl cyclase activity, suggesting that this compound has irreversible binding characteristics. In addition, SB-(+)-273779 antagonized CGRP-mediated 1) stimulation of intracellular Ca(2+) in recombinant CGRP receptors in HEK-293 cells, 2) inhibition of insulin-stimulated [(14)C]deoxyglucose uptake in L6 cells, 3) vasodilation in rat pulmonary artery, and 4) decrease in blood pressure in anesthetized rats. SB-(+)-273779 tested at 3 microM had no significant affinity for calcitonin, endothelin, angiotensin II, and alpha-adrenergic receptors under standard ligand binding assays. SB-(+)-273779 also did not inhibit forskolin and pituitary adenylate cyclase-activating polypeptide. These results suggest that SB-(+)-273779 is a valuable tool for studying CGRP-mediated functional responses in complex biological systems.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

In vitro and in vivo characterization of intrinsic sympathomimetic activity in normal and heart failure rats.

Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats.

The myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF) rats.

-Responsiveness to beta-adrenergic stimulation is reduced in the failing human myocardium. This results principally from reduced beta-adrenergic receptor (betaAR) density, elevated beta-adrenergic receptor kinase 1 (betaARK1) levels, and functional uncoupling of remaining receptors. The temporal nature of changes in the human myocardial beta-adrenergic system relative to onset of symptomatic heart failure (HF) has been difficult to discern. A relatively new model of HF, the spontaneously hypertensive heart failure (SHHF) rat spontaneously and reproducibly develops left ventricular hypertrophy (LVH) and progresses to HF, thus enabling longitudinal studies to examine the cellular and molecular bases for hypertension-induced cardiac hypertrophy and subsequent HF. The purpose of this study was to examine age-dependent changes in the betaAR system in this model. Lean male SHHF rats at 3, 7, 14, and 20 months were compared with age-matched Sprague-Dawley (SD) control rats ([C]; 4 animals/group). At all ages the SHHF rats had elevated blood pressures and left ventricular end-diastolic pressure relative to the SD control rats (P<0.05). Compared with age-matched SD control rats, LVH was evident by 3 months in SHHF rats; 20-month-old SHHF rats had significantly greater LVH compared with the other SHHF rat groups. beta-adrenergic responsiveness (maximal heart rate to isoproterenol) was reduced only in 20-month-old SHHF rats. betaARK1 protein levels and activity were elevated at 14 months (162+/-10% and 195+/-20% C, respectively), and betaARK1 protein remained elevated at 20 months (140+/-14% C). In contrast, G protein-coupled receptor kinase 5, a second receptor kinase in the heart, remained unchanged at all ages. betaAR density did not change with age in the SD control rats and was similar in the SHHF rats until 20 months of age when the receptor number was reduced (30+/-1%). These data indicate that cardiac dysfunction is coincident with reduced betaAR density. Importantly, cardiac dysfunction was preceded by elevated betaARK1 levels and activity, thus suggesting that betaARK1 may be a precipitating factor in the transition from hypertension-induced compensatory cardiac hypertrophy to HF. Furthermore, these results indicate that the SHHF rat is a powerful model for use in examination of the mechanisms involved in alterations of beta-adrenergic signaling that occur in human HF.

Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury.

BACKGROUND AND PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that rapidly upregulates in the brain after injury. The present study was designed to explore the pathophysiological significance of brain TNF-alpha in the ischemic brain by systematically evaluating the effects of lateral cerebroventricular administration of exogenous TNF-alpha and agents that block the effects of TNF-alpha on focal stroke and by examining the potential direct toxic effects of TNF-alpha on cultured neurons to better understand how TNF-alpha might mediate stroke injury. METHODS: TNF-alpha (2.5 or 25 pmol) was administered intracerebroventricularly to spontaneously hypertensive rats 24 hours before permanent or transient (80 minutes and 160 minutes) middle cerebral artery occlusion (MCAO). Animals were examined 24 hours later for neurological deficits and ischemic hemisphere necrosis and swelling. In some of these studies, neutralizing anti-TNF-alpha monoclonal antibody (mAb) (60 pmol) was injected intracerebroventricularly 30 minutes before exogenous TNF-alpha (25 pmol). In addition, the effects of blocking endogenous TNF-alpha on permanent focal ischemic injury were determined with the use of either mAb (60 pmol) or soluble TNF receptor I (sTNF-RI) (0.3 or 0.7 nmol) administered intracerebroventricularly 30 minutes before and 3 and 6 hours after MCAO. Finally, the direct neurotoxic effects of TNF-alpha were studied in cultured rat cerebellar granule cells exposed to TNF-alpha (10 to 2000 U/mL for 6 to 24 hours), and neurotransmitter release, glutamate toxicity, and oxygen radical toxicity were studied. RESULTS: TNF-alpha increased the percent hemispheric infarct induced by permanent MCAO in a dose-related manner from 13.1 +/- 1.3% (vehicle) to 18.9 +/- 1.7% at 2.5 pmol (P < .05) and 27.1 +/- 1.3% at 25 pmol (P < .0001). The high dose of TNF-alpha increased ischemia-induced forelimb deficits from 1.6 +/- 0.2 to 2.3 +/- 0.2 (P < 0.1). TNF-alpha (2.5 pmol) also increased the infarction induced by 80 or 160 minutes of transient MCAO from 1.9 +/- 0.9% to 4.3 +/- 0.4% (P < .01) and from 14.2 +/- 1.3% to 21.6 +/- 2.2% (P < .05), respectively. The exacerbation of infarct size, swelling, and neurological deficit after exogenous TNF-alpha was reversed by preinjection of 60 pmol mAb. Blocking endogenous TNF-alpha also significantly reduced focal ischemic brain injury. Treatment with 60 pmol mAb before and after permanent MCAO significantly reduced infarct size compared with control (nonimmune) antibody treatment by 20.2% (P < .05). Reduced brain infarction also was produced by brain administration of 0.3 nmol (decreased 18.2%) or 0.7 nmol (decreased 26.1%, P < .05) sTNF-RI before and after focal stroke. The intracerebroventricular administration of TNF-alpha or sTNF-RI did not alter brain or body temperature, blood gases or pH, blood pressure, blood glucose, or general blood chemistry. In cultured cerebellar granule cells, the application of TNF-alpha did not directly affect neurotransmitter release or glutamate or oxygen free radical toxicity. CONCLUSIONS: These studies demonstrate that exogenous TNF-alpha exacerbates focal ischemic injury and that blocking endogenous TNF-alpha is neuroprotective. The specificity of the action(s) of TNF-alpha was demonstrated by antagonism of its effects with specific anti-TNF-alpha tools (ie, mAb and sTNF-RI). TNF-alpha toxicity does not appear to be due to a direct effect on neurons or modulation of neuronal sensitivity to glutamate or oxygen radicals and apparently is mediated through nonneuronal cells. These data suggest that inhibiting TNF-alpha may represent a novel pharmacological strategy to treat ischemic stroke.

Identification, characterization, and functional role of phosphodiesterase type IV in cerebral vessels: effects of selective phosphodiesterase inhibitors.

The role of the phosphodiesterase type IV isozyme (PDE IV) in the regulation of cerebrovascular tone was investigated in the canine basilar artery in vitro and in vivo. The PDE isozymes extracted from the canine basilar artery were isolated by diethylaminoethanol (DEAE)-Sepharose affinity chromatography and identified based on sensitivity to isozyme-selective PDE inhibitors. [3H]cAMP hydrolysis was observed in one major and one minor peak of activity. The predominant peak was inhibited by the addition of cGMP (25%), siguazodan (26%), rolipram (39%), and the combination of siguazodan and rolipram (95%). Selective PDE IV inhibitors BRL 61063, rolipram, and denbufylline were equieffective inhibitors of [3H]-ccAMP hydrolysis mediated by PDE IV isolated from the canine basilar artery [concentrations producing 50% inhibition (IC50S) = 0.21 +/- 0.05 microM, 0.67 +/- 0.23 microM, and 0.73 +/- 0.16 microM, respectively]. In precontracted isolated ring segments of the canine basilar artery, selective PDE IV inhibitors produced potent and complete relaxation (IC50S < 150 nM). In contrast, zaprinast (a selective PDE V inhibitor) and siguazodan (a selective PDE III inhibitor) produced only weak relaxation of the basilar artery (IC50S = 4.5 microM and > 10 microM, respectively). Vasorelaxation produced by PDE IV inhibitors was not altered by removing the endothelium, 1-NAME, or adenosine receptor antagonism. In a canine model of acute cerebral vasospasm, all three selective PDE IV inhibitors reversed basilar artery spasm produced by autologous blood without altering mean arterial blood pressure. In contrast, prolonged treatment with BRL 61063 failed to alter the development of basilar spasm in the two hemorrhage canine models of chronic cerebral vasospasm. Denbufylline-induced relaxation in vitro was also significantly impaired in basilar arteries obtained from the model of chronic vasospasm. In conclusion, PDE IV appears to be the predominant isozyme regulating vascular tone mediated by cAMP hydrolysis in cerebral vessels. In addition, vasorelaxation modulated by PDE IV is compromised in chronic cerebral vasospasm associated with subarachnoid hemorrhage.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Differential calcitonin gene-related peptide (CGRP) and amylin binding sites in nucleus accumbens and lung: potential models for studying CGRP/amylin receptor subtypes.

Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[125I]iodohistidyl10)humanCGRP alpha ([125I]hCGRP alpha) binding (IC50 = 0.4-7.7 nM), which was displaced by hCGRP8-37 alpha with equally high affinity (IC50 = 0.4-7.3 nM). High-affinity binding for [125I]Bolton-Hunter human amylin ([125I]BH-h-amylin) was also observed in these tissues (IC50 = 0.2-6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC50 = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin8-37 competed for [125I]hCGRP alpha binding with higher affinity (IC50 = 5.4 nM) compared with [125I]BH-h-amylin binding (IC50 = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC50 values for rat amylin8-37 were 117 and 12 nM against [125I]hCGRP alpha and [125I]BH-h-amylin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.

The consequences of cerebral ischemia were studied in three different strains (BDF, CFW, and BALB/C) of mice. The different strains exhibited significant differences in susceptibility to 24-h focal ischemia. Following middle cerebral artery occlusion (MCAO), infarct volumes (mm3) were 5 +/- 3 in BDF, 15 +/- 5 in CFW, and 23 +/- 3 in BALB/C mice (p < 0.05). MCAO plus ipsilateral common carotid artery occlusion (CCAO) resulted in infarct volumes of 15 +/- 9 in BDF, 38 +/- 10 in CFW, and 72 +/- 12 in BALB/C mice (p < 0.05). In addition, MCAO plus CCAO produced death by 24 h in 42% of CFW and 67% of BALB/C mice, but not in any BDF mice (p < 0.05). CCAO alone produced multifocal hemispheric infarctions in 36% of BALB/C mice but not in the other two strains. Brains of all mouse strains subjected to sham surgery were free of any ischemic injury. Arterial blood pressures, blood gases, and blood cell profiles were relatively similar for the three mouse strains. However, carbon black studies of the cerebrovascular anatomy revealed an incomplete circle of Willis (i.e., a significant decrease in the frequency of patent posterior communicating arteries) for BALB/C compared with BDF mice (p < 0.05), with CFW mice being intermediary. Based on these anatomical data, BALB/C mice also were evaluated following transient global brain ischemia produced by bilateral CCAO. BALB/C mice exhibited a > 85% reduction in cortical microvascular perfusion and EEG power within 1 min of bilateral CCAO. Also, hippocampal neuronal CA1 damage and mortality over 7 days were related to the duration of global brain ischemia (p < 0.05). These data demonstrate a significant difference between mouse strains in their sensitivity to cerebral ischemia that appears to be related, at least in part, to the functional vascular anatomy at the level of the posterior communicating arteries. In particular, we point out the potential usefulness of BALB/C mice as a sensitive and reproducible model of focal and global ischemia.

Antithrombotic effects of a platelet fibrinogen receptor antagonist in a canine model of carotid artery thrombosis.

BACKGROUND AND PURPOSE: Platelet-fibrin thrombi in the lumen of atherostenotic carotid arteries may underlie transient ischemic attacks and cerebral infarction. For this reason, we investigated the antiplatelet and antithrombotic effects of a novel and potent platelet fibrinogen receptor (glycoprotein IIb/IIIa) antagonist (SK&F 106760). METHODS: The effects of 0.1-3.0 mg/kg i.v. SK&F 106760 on platelet aggregation were examined ex vivo in canine platelet-rich plasma (n = 20). In addition, the antithrombotic effects of SK&F 106760 were compared with those of aspirin in an acute canine model of extracranial carotid artery thrombosis with high-grade stenosis. Sham-operated (n = 4), vehicle-treated (n = 6), SK&F 106760-treated (n = 8), aspirin-treated (n = 9), and SK&F 106760+aspirin-treated (n = 5) dogs were examined. RESULTS: The intravenous administration of SK&F 106760 caused a dose-related inhibition of ex vivo platelet aggregation. In the carotid artery thrombosis model, an occlusive thrombus formed at stenotic sites in the region of the carotid bifurcation. The thrombogenic process caused a progressive reduction in carotid blood flow and reduced the cortical microvascular perfusion and electroencephalographic power. Based on nuclear magnetic resonance spectroscopy, the occlusive events depleted the stores of high-energy phosphates (adenosine triphosphate and phosphocreatine) and increased the lactate concentration in the forelimb somatosensory area of the parietal cortex. In this model, the administration of 1 mg/kg i.v. SK&F 106760 prevented thrombosis of the stenotic carotid artery. Consequently, neurophysiological, cerebral hemodynamic, and metabolic parameters were all improved significantly in the SK&F 106760-treated group. No dog receiving SK&F 106760 reoccluded during the 1-hour posttreatment observation period. In contrast, thrombosis of the carotid artery was associated with neurophysiological deterioration in six of the nine dogs treated with 5 mg/kg i.v. aspirin. Both spontaneous and evoked (increased carotid stenosis) aspirin-resistant thrombosis were abolished by SK&F 106760 treatment. CONCLUSIONS: These results suggest that antagonism of fibrinogen binding to platelet glycoprotein IIb/IIIa (the final common pathway for aggregation) may represent a new and more effective antithrombotic approach to the treatment of cerebral transient ischemic attacks and infarction associated with extracranial carotid artery disease.

Distinction of NPY receptors in vitro and in vivo. I. NPY-(18-36) discriminates NPY receptor subtypes in vitro.

We studied the possibility of multiple neuropeptide Y (NPY) receptor subtypes. NPY-stimulated Ca2+ mobilization in human erythroleukemia (HEL) cells was used to screen a number of NPY analogues. The potencies of three of these analogues [peptide YY (PYY), [D-Tyr-36]NPY, and NPY-(18-36)] were compared with that of NPY in the following model systems: Ca2+ mobilization and inhibition of adenosine 3',5'-cyclic monophosphate accumulation in HEL cells, potentiation of vasoconstriction in the isolated rabbit ear artery, reduction of cutaneous microvascular perfusion in the rat digit, and inhibition of [3H]serotonin release in rat brain. In each of the five models, PYY was a full agonist that exhibited a similar or slightly higher potency than NPY, whereas [D-Tyr-36]NPY and NPY-(18-36) were partial agonists with lower potencies: NPY-(18-36) had a lower potency and efficacy than [D-Tyr-36]NPY in HEL cells and the rabbit ear artery, but was more effective than [D-Tyr-36]NPY for constricting cutaneous microvasculature and inhibiting serotonin release. Because of its weak partial agonism, we also tested NPY-(18-36) as an antagonist of NPY-stimulated Ca2+ mobilization in HEL cells. NPY-(18-36) shifted the NPY concentration-response curve to the right with a KB affinity value of 297 nM. In summary, [D-Tyr-36]NPY and NPY-(18-36) are partial agonists, the relative potency of which varies between systems. These data demonstrate the presence of multiple NPY receptor subtypes. We propose a modified classification scheme of NPY receptor subtypes.

Effect of endothelin on cortical microvascular perfusion in rats.

We used laser-Doppler flowmetry to study the effects of endothelin-1 on local cortical microvascular perfusion and resistance in 29 pentobarbital-anesthetized rats. Intravenous administration of 10-300 pmol endothelin-1 reduced arterial blood pressure and microvascular resistance and increased microvascular perfusion. However, intracarotid administration of low doses of endothelin-1 increased microvascular perfusion and reduced microvascular resistance and arterial blood pressure, whereas high doses (greater than or equal to 300 pmol) reduced microvascular perfusion and increased microvascular resistance and arterial blood pressure. Only the high dose/low flow response was associated with attenuation of the electrocorticogram. The low dose/high flow and high dose/low flow responses to endothelin-1 were not altered by blockade of muscarinic and adrenergic receptors. In addition, systemic metabolic changes (arterial pH, PaCO2, PaO2, and plasma glucose concentration) did not account for the cerebrovascular effects of endothelin-1. Platelet hyperaggregability also did not appear to be a causative factor in the high dose/low flow response to endothelin-1. In fact, ex vivo rat platelet aggregation was inhibited by intracarotid administration of 300 pmol endothelin-1. In conclusion, the cerebral vasculature exhibits extreme sensitivity to the vasodilator properties of endothelin-1 at low doses. The ischemic vasoconstrictor effects observed at high doses implicate endothelin-1 as an important mediator of cerebral vasospasm and/or postischemic hypoperfusion.

Urinary cannabinoids in monitoring abstinence in a drug abuse treatment program.

Urine specimens from 162 adolescents entering a drug treatment program were tested for cannabinoids using a photometric immunoassay (EMITst) and thin-layer chromatography (TLC). The EMITst has a cutoff point of detection 25 ng/mL or less of 9-carboxy-tetrahydrocannabinol. When reported as positive, both tests appeared to be reliable. There were two false-positive EMITst results and three false-positive TLC results in the 67 urine specimens that did not contain cannabinoids. When reported as negative, however, the EMITst with its 100 ng/mL cutoff failed to detect almost 40% of all cannabinoid-positive specimens. Of the 65 chronic marijuana smokers in the present study who stated that they had smoked within two days of their admission into the treatment facility, 17 (26%) went undetected by the 100 ng/mL cutoff used by the EMITst method. In clinical settings such as drug treatment programs, tests for urinary cannabinoids should use a detection threshold at 20 ng/mL or less.

Evaluation of immunoassays for cannabinoids in urine.

A comparison was made of several cannabinoid urine assays. Two hundred randomly selected urine specimens were initially screened by two enzyme immunoassays (EMIT-st and EMIT-d.a.u.) and a radioimmunoassay (Abuscreen RIA). Selected specimens found positive by any of these methods were further analyzed by gas-liquid chromatography with flame ionization detection (GLC/FID), gas chromatography/mass spectrometry (GC/MS), and an experimental RIA from Research Triangle Institute (RTI RIA). The GLC/FID method gave confirmations in 69 to 92% of the samples, depending on the method used and the cut-off employed. GC/MS confirmed 98% of the EMIT and RIA positives using a low cut-off (20 ng/mL). All RIA positives at 100 ng/mL were confirmed by GC/MS. There was complete agreement between the RTI RIA and the EMIT assays, but not with the Abuscreen RIA at the 100 ng/mL cut-off. The study illustrates that care must be exercised in establishing assay cut-offs and the designation of false positive results.