Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice

BACKGROUND: Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS: We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS: PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.     

Serotonin is involved in the ACTH-induced reversal of hemorrhagic shock in anesthetized rats

In a rat model of volume-controlled hemorrhagic shock (mean arterial pressure = 20-24 mm Hg) causing the death of all saline-treated animals within 30 min, the i.v. bolus injection of ACTH-(1-24) (160 micrograms/kg) produced an almost complete and sustained reversal of the shock condition, with recovery of arterial blood pressure, pulse pressure and respiratory rate, and with 100% survival at the end of the experiment (2 h). The serotonin-depleting agent p-chlorophenylalanine (316 mg/kg i.p., administered 66-70 h before hemorrhage) almost completely prevented the effect of ACTH. The 5-HT1/5-HT2 receptor antagonist, methysergide, prevented the effect of ACTH completely when injected i.v. (5 mg/kg), but only in part when injected into a brain ventricle (i.c.v.) (15 micrograms/rat); the 5-HT2 antagonist, ketanserin, prevented the effect of ACTH completely when injected i.c.v. (1.5 micrograms/rat), but only in part when injected i.v. (0.5 mg/kg); the 5-HT3 antagonist, MDL 72222, largely prevented the effect of ACTH when injected i.c.v. (10 micrograms/rat), but had no influence at all when injected i.v. (3 mg/kg); finally, the 5-HT4 antagonist, GR 125487, had no effect when injected i.v. (5 micrograms/kg) or when injected i.c.v. (30 ng/rat). Overall, these data indicate that both CNS and peripheral serotonin play an important role in the complex mechanism of the ACTH-induced hemorrhagic shock reversal.     

Recombinant lys-plasminogen given before, but not after, recombinant tissue-type plasminogen activator markedly improves coronary thrombolysis in dogs: relationship of thrombolytic efficacy with parameters of fibrinolysis

Recombinant tissue-type plasminogen activator (rt-PA) administration rapidly restores blood flow in thrombosed coronary arteries, but coronary arteries often reocclude after initial thrombolysis. This occurs because of the short half-life of rt-PA and rapid increase in plasminogen activator inhibitor (PAI-1) and alpha2-antiplasmin levels in plasma. We hypothesized that administration of lys-plasminogen, which binds to fibrin with 10 times greater affinity and results in a loose fibrin structure (as compared with native glu-plasminogen), before rt-PA would enhance the thrombolytic efficacy of rt-PA and modulate parameters of fibrinolysis. To examine this hypothesis, dogs with electrically induced stable thrombus in the left anterior descending coronary artery (LAD) were treated with saline (group A, n = 9) or lys-plasminogen (group B, 2 mg/kg, n = 5), followed 10 min later by rt-PA (1 mg/kg in 20 min). Four other dogs with occlusive LAD thrombus were first given rt-PA, followed by lys-plasminogen (2 mg/kg) 50 min later (group C). Lys-plasminogen given before rt-PA restored flow in all dogs in 14 +/- 4 min (vs. 22 +/- 9 min in group A, p < 0.05), continuing > 2 h (vs. 41 +/- 15 min in group A, p < 0.02). Lys-plasminogen given after rt-PA did not potentiate the effect of rt-PA. Plasma t-PA antigen concentrations were highest in group B dogs at 2 h after rt-PA infusion. PAI-1 and alpha2-antiplasmin plasma levels were suppressed in all dogs receiving lys-plasminogen whether it was given before or after rt-PA. Therefore, lys-plasminogen given before rt-PA markedly potentiates the effect of rt-PA and alters the parameters of fibrinolysis. In contrast, lys-plasminogen given after rt-PA does not influence the thrombolytic effect of rt-PA, whereas it suppresses PAI-1 and alpha2-antiplasmin levels in plasma. This study also suggests that binding of plasminogen to the clot is more important than the plasma levels of PAI-1 and alpha2-antiplasmin.     

Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals

BACKGROUND: Ischemia with or without reperfusion induces the release of diverse products from monocytes, including cytokines such as interleukin-1 (IL-1). To determine whether these phenomena modulate fibrinolysis and potentially exacerbate impairment of the macrocirculation, microcirculation, or both, we characterized the effects of IL-1 on the expression of fibrinolytic system and matrix proteins in rat cardiac microvascular endothelial cells (CMECs). METHODS AND RESULTS: Confluent CMECs were exposed to IL-1 in serum-free medium for 24 hours, and cell-conditioned medium was assayed for plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of plasminogen activators, and for type 1 collagen with Western blotting. IL-1 (2 ng/mL) specifically increased the accumulation of PAI-1 (4.4 +/- 0.6-fold; mean +/- SD; n = 9) without affecting tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA) levels, which remained unchanged. IL-1 increased the accumulation of collagen in conditioned media by 3.5 +/- 0.7-fold (n = 6). Conversely, the accumulation of both PAI-1 and collagen induced by IL-1 was inhibited with an IL-1 receptor antagonist (200 ng/mL; n = 6) and with cycloheximide (10 micrograms/mL; n = 6), implying that protein synthesis was a requirement for the effect. To determine whether the IL-1 effect was mediated by induction of oxygen-centered free radical production, known to be induced by IL-1, we exposed the cells to the hydroxyl radical scavenger tetramethylthiourea (10 mmol/L) and observed abolition of the IL-1-induced increase in the expression of PAI-1 and collagen (n = 6). Conversely, superoxides (generated with 10 mU/mL xanthine oxidase plus 0.6 mmol/L hypoxanthine, and 100 mumol/L hydrogen peroxide) induced the accumulation of PAI-1 and collagen (n = 6). IL-1 (1 microgram/kg body wt) and lipopolysaccharide (50 micrograms/kg body wt) administered in vivo increased PAI-1 protein in rat hearts as detected with Western blotting and PAI-1 immunostaining of rat heart microvessels, indicating the effects delineated in vitro were paralleled by effects in vivo. CONCLUSIONS: These results indicate that IL-1-induced oxygen-centered free radicals stimulate elaboration of PAI-1 and collagen by CMECs. Accordingly, microvascularly mediated inhibition of fibrinolysis may predispose to the persistence of microvascular thrombi, thereby contributing to impaired microcirculatory function, the no-reflow phenomenon, and cardiac dysfunction after ischemia and reperfusion.     

Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance. Studies with thrombi generated in the Chandler loop

To investigate the potential role of plasminogen activator inhibitor-1 (PAI-1), which is released from the alpha-granules of activated platelets, in thrombolysis resistance, we employed a model (the "Chandler loop") that mimics the formation of arterial thrombi in vivo and that can be manipulated in terms of rheological parameters and composition of blood cells. Light and electron microscopy revealed that the distribution of blood cells in Chandler thrombi is polarized, as it is in arterial thrombi, resulting in platelet-rich "white heads" and red blood cell-rich "red tails.". Resistance toward tissue-type plasminogen activator (TPA)-mediated thrombolysis parallels the presence of platelets that are fully activated in this system. We demonstrate that the PAI-1 released by the alpha-granules is preferentially retained within the thrombus and that the concentration of PAI-1 antigen is higher in the head than in the tail of the thrombus. The relative thrombolysis resistance of the heads of Chandler thrombi can be largely abolished by inclusion of an anti-PAI-1 monoclonal antibody that blocks that inhibitory activity of PAI-1 toward TPA. We propose that PAI-1, released from activated platelets, plays a key role in thrombolysis resistance and/or reocclusion after thrombolytic therapy. This is due to binding of PAI-1 to polymerized fibrin within the thrombus, followed by inhibition of TPA-mediated fibrinolysis.     

Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues

Rats (Sprague-Dawley), submitted to a mechanical noxious stimulus (paw pressure), were tested to determine 1) the antinociceptive effects of p.o. (200, 400 and 800 mg/kg), i.v. (50, 100, 200 and 300 mg/kg) and intrathecal (i.t.) (100 and 200 micrograms/rat) administrations of paracetamol; 2) the influence of i.t. administered tropisetron, a 5-hydroxytryptamine3 (5-HT3) receptor antagonist (0.5, 1 or 10 micrograms/rat) on paracetamol-induced antinociception; 3) the influence of indomethacin (25 mg/kg s.c.), naloxone (10 micrograms/rat i.t.) and yohimbine (1 mg/kg i.v.) on the effect of paracetamol (200 mg/kg i.v.) to determine the involvement of prostaglandins, opioids and alpha-2 adrenoceptors. The displacement by paracetamol of radioligand binding to various receptors was also investigated. Paracetamol induced a significant antinociceptive effect after p.o., i.v. and i.t. administration. A total inhibition of the effect of paracetamol, administered p.o. or i.t., occurred at the dose of 0.5 microgram/rat of tropisetron, whereas 10 micrograms/rat of this antagonist was needed to totally inhibit the action of i.v. administered paracetamol. Indomethacin, naloxone and yohimbine failed to modify paracetamol antinociceptive action. In vitro studies failed to show any binding of paracetamol to 5-HT3 and several other receptors and to 5-HT uptake sites. It is concluded that paracetamol has a central antinociceptive effect, based on an indirect involvement of spinal 5-HT3 receptors.     

Antithrombotic effects in a rat model of aspirin-insensitive arterial thrombosis of desethyl KBT-3022, the main active metabolite of a new antiplatelet agent, KBT-3022

The antithrombotic effect of desethyl KBT-3022, which is the main active metabolite of the new antiplatelet agent, KBT-3022 (ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl] pyrrol-1-ylacetate; a cyclooxygenase inhibitor), was determined using a photochemically induced arterial thrombosis model in the rat femoral artery. Pretreatment with desethyl KBT-3022 (0.1, 0.3 and 1 mg/kg, i.v.) prolonged the time required to achieve thrombotic occlusion in the femoral artery and inhibited collagen-induced platelet aggregation in whole blood ex vivo, each in a dose-dependent manner. In all 6 rats used, particularly at the highest dose (1 mg/kg, i.v.) tested, cyclic variations in blood flow were hardly ever observed and complete cessation of blood flow did not occur during the 30-min observation time. BM-13505 (1, 3 and 10 mg/kg, i.v.), a thromboxane A2 receptor antagonist, also prolonged the time to occlusion, but cyclic variations in blood flow did occur. On the other hand, aspirin (10 and 30 mg/kg, i.v.) had little effect in terms of preventing thrombosis, although it inhibited collagen-induced platelet aggregation to the same extent as did desethyl KBT-3022. Desethyl KBT-3022 inhibited the thrombin-induced aggregation of washed platelets in a concentration-dependent manner (1-40 microM), whereas aspirin and BM-13505 did not. These findings suggest that the potent antithrombotic effect of desethyl KBT-3022 may be attributable in part to its additional ability to inhibit thrombin-induced platelet aggregation. Accordingly, thromboxane A2 and thrombin may be important thrombotic mediators in this rat model.     

Modulation of plasminogen activator inhibitor type-1 biosynthesis in vitro and in vivo with oligo(nucleoside phosphorothioate)s and related constructs

Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.     

Evidence that histamine is the principal pharmacological component of venom from an Australian wolf spider (Lycosa godeffroyi)

Wolf spiders are common throughout Australia and have been known to cause severe reactions in both animals and humans. However, little work has been done on the pharmacological activity of Australian lycosids. The purpose of this study was to obtain a preliminary pharmacological profile of the venom from an Australian wolf spider (Lycosa godeffroyi). The venom caused dose-dependent contractions of guinea-pig isolated ileum (1-4 microg/ml), endothelium-dependent relaxation in rat isolated aortae (10 microg/ml), a decrease in mean arterial blood pressure in the anaesthetised rat (100 microg/kg, i.v.) and an increase in insufflation pressure in the anaesthetised guinea-pig (50 microg/kg, i.v.). All of these responses were significantly inhibited by the H1-receptor antagonist mepyramine at concentrations that selectively inhibited responses to histamine. Venom (5 microg/ml) caused a decrease in twitch height of the rat stimulated (0.3 msec, 0.2 Hz, 100 V) vas deferens (prostatic segment). A fluorometric assay for histamine detected a concentration of 44.5 ng/microg venom protein. It appears that the in vitro and in vivo activity of L. godeffroyi venom observed in the present study is due to the presence of histamine.     

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound. 2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg-1, p.o., 0.03 to 1 mg kg-1, i.p. and 0.1 to 1 microgram/rat, i.c.v.) increased urine output and urinary excretion of Na+, K+ and Cl- and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats. 3. NT (0.1 microgram/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 microgram/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 microgram/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis. 4. The NO synthesis inhibitor N(1)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg-1, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg-1, i.p.) but not D-arginine (1 g kg-1, i.p.) restored the SR 48692-dependent increase in diuresis, L-NAME had no effect on furosemide-stimulated diuresis. 5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine. 6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692. 7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg-1, i.p.) stimulated urine output. 8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na+, K+ and Cl-.     

Diagnostic concordance of telecytology and conventional cytology for evaluating breast aspirates

We examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 microg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.     

Endomorphin 1 and 2 have vasodepressor activity in the anesthetized mouse

The endogenous peptides endomorphin 1 and 2 are newly discovered, potent, selective mu-opioid receptor agonists. In the present study, we investigated responses to the endomorphin peptides in the systemic vascular bed of the anesthetized mouse. Endomorphin 1 and 2 induced dose-related decreases in mean arterial pressure when injected in doses of 3-100 nmol/kg i.v. Mean arterial pressure decreased 14 +/- 4, 23 +/- 4, and 42 +/- 5 mm Hg at the 10, 30, and 100 nmol/kg doses, respectively, of endomorphin 1 (n = 5-7; p < 0.05), and similar changes were observed in response to endomorphin 2. In terms of relative vasodepressor activity, endomorphin 1 and 2 were about equipotent and about threefold more potent than the mu-opioid selective agonist PL017 in decreasing mean arterial pressure; all three peptides decreased heart rate. The time-course of the vasodepressor responses to endomorphin 1 and 2 were similar in rate of onset and decay. Vasodepressor responses to endomorphin 1 and 2 and PL017 but not to nociceptin were inhibited by the opioid receptor antagonist naloxone in a dose of 2 mg/kg i.v. When compared in the mouse and rat, the relative decreases in systemic arterial pressure in response to i.v. injections of endomorphin 1 and 2 did not differ greatly. However, the duration of the vasodepressor response was significantly longer in the rat. These results demonstrate that endomorphin 1 and 2 have significant, naloxone-sensitive, vasodepressor activity in the mouse.     

Arachidonic acid and its metabolites are involved in the expression of morphine dependence in guinea-pig isolated ileum

This study was undertaken to determine if the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), is a competitive antagonist of muscarinic receptors in vivo. Cats were anesthetized with pentobarbital (36 mg/kg, i.p.). Five peripheral muscarinic responses were characterized based on their sensitivity to intravenous administration of atropine (1-100 microg/kg), pirenzepine (1-100 microg/kg) or gallamine (30-3000 microg/kg) as follows: (1) muscarinic ganglionic transmission through the superior cervical ganglion to the nictitating membrane (M1), (2) electrically elicited vagal bradycardia (M2), (3) neurally evoked sudomotor responses (M3; non-endothelial), (4) basal pupil tone in sympathectomized cats (M3; non-endothelial) and (5) methacholine-induced depression of arterial blood pressure (M3; endothelial). Additional groups of animals were administered L-NAME (50 mg/kg, i.v.) to determine if this agent would alter activation of these muscarinic systems. L-NAME was devoid of effect on responses elicited by stimulation of muscarinic M1, M2 and M3 (non-endothelial) receptors. In contrast, L-NAME significantly reduced the depressor responses to i.v. methacholine (M3; endothelial), as did its non-alkyl ester congener, L-NA (NG-nitro-L-arginine; 25 mg/kg, i.v.). These results support the conclusion that although L-NAME inhibits synthesis of nitric oxide in vascular endothelial cells, it is not a generalized muscarinic receptor antagonist in vivo.     

Effect of a selective 5-HT reuptake inhibitor in combination with 5-HT1A and 5-HT1B receptor antagonists on extracellular 5-HT in rat frontal cortex in vivo

1. Selective 5-hydroxytryptamine (5-HT; serotonin) reuptake inhibitors (SSRIs) cause a greater increase in extracellular 5-HT in the forebrain when the somatodendritic 5-HT1A autoreceptor is blocked. Here, we investigated whether blockade of the terminal 5-HT1B autoreceptor influences a selective 5-HT reuptake inhibitor in the same way, and whether there is an additional effect of blocking both the 5-HT1A and 5-HT1B autoreceptors. 2. Extracellular 5-HT was measured in frontal cortex of the anaesthetized rat by use of brain microdialysis. In vivo extracellular recordings of 5-HT neuronal activity in the dorsal raphe nucleus (DRN) were also carried out. 3. The selective 5-HT reuptake inhibitor, paroxetine (0.8 mg kg-1, i.v.), increased extracellular 5-HT about 2 fold in rats pretreated with the 5-HT1A receptor antagonist, WAY100635. When administered alone neither paroxetine (0.8 mg kg-1, i.v.) nor WAY100635 (0.1 mg kg-1, i.v.) altered extracellular 5-HT levels. 4. Paroxetine (0.8 mg kg-1, i.v.) did not increase 5-HT in rats pretreated with the 5-HT1B/D receptor antagonist, GR127935 (1 mg kg-1, i.v.). GR127935 (1 and 5 mg kg-1, i.v.) had no effect on extracellular 5-HT when administered alone. 5. Interestingly, paroxetine (0.8 mg kg-1, i.v.) caused the greatest increase in 5-HT (up to 5 fold) when GR127935 (1 or 5 mg kg-1, i.v.) was administered in combination with WAY100635 (0.1 mg kg-1, i.v.). Administration of GR127935 (5 mg kg-1, i.v.) plus WAY100635 (0.1 mg kg-1, i.v.) without paroxetine, had no effect on extracellular 5-HT in the frontal cortex. 6. Despite the lack of effect of GR127935 on 5-HT under basal conditions, when 5-HT output was elevated about 3 fold (by adding 1 microM paroxetine to the perfusion medium), the drug caused a dose-related (1 and 5 mg kg-1, i.v.) increase in 5-HT. 7. By itself, GR127935 slightly but significantly decreased 5-HT cell firing in the DRN at higher doses (2.0-5.0 mg kg-1, i.v.), but did not prevent the inhibition of 5-HT cell firing induced by paroxetine. 8. In summary, our results suggest that selective 5-HT reuptake inhibitors may cause a large increase in 5-HT in the frontal cortex when 5-HT autoreceptors on both the somatodendrites (5-HT1A) and nerve terminals (5-HT1B) are blocked. This increase is greater than when either set of autoreceptors are blocked separately. The failure of a 5-HT1B receptor antagonist alone to enhance the effect of the selective 5-HT reuptake inhibitor in our experiments may be related to a lack of tone on the terminal 5-HT1B autoreceptor due to a continued inhibition of 5-HT cell firing. These results are discussed in relation to the use of 5-HT autoreceptor antagonists to augment the antidepressant effect of selective 5-HT reuptake inhibitors.     

Do kinins mediate cardioprotective and renoprotective effects of cilazapril in spontaneously hypertensive rats with renal ablation?

1. We assessed the potential of the kallikrein-kinin system in mediating the cardioprotective and renoprotective effects of an angiotensin-converting enzyme inhibitor (ACEI), cilazapril (CIL) in rats with renal ablation. 2. Eight week old spontaneously hypertensive rats (SHR) were subjected to 5/6 nephrectomy. One week after the operation, the rats were divided into 5 groups: (i) vehicle; (ii) CIL 1 mg/kg per day per os (p.o.); (iii) Hoe140 (HOE) 70 mu g/kg per day given intraperitoneally (i.p.); (iv) CIL 1 mg/kg per day p.o. plus HOE 7 mu g/kg per day i.p.; (v) CIL 1 mg/kg per day p.o. plus HOE 70 mu g/kg per day i.p. The treatment lasted for 4 weeks. 3. CIL alone significantly reduced systolic blood pressure, urinary protein excretion, heart weight and serum creatinine level. HOE alone did not induce any significant changes in these parameters. CIL in combination with HOE (7 or 70 mu g/kg per day) did not induce any changes in these parameters, in addition to those associated with the effects of CIL alone. 4. These results indicate that the kallikrein-kinin system might not play a major role in the cardioprotective and renoprotective effects of ACE inhibitors in the rat remnant kidney model of chronic renal failure.     

Antiplatelet effect of FK633, a platelet glycoprotein IIb/IIIa antagonist, on thrombus formation and vascular patency after thrombolysis in the injured hamster carotid artery

The antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) IIb/IIIa receptor, were studied, IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 microM adenosine diphosphate (ADP) was 5.4 x 10(-7) M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 +/- 1.1 min, mean +/- S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1, 0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i.v. by an implanted osmotic pump for 3, 7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.     

Central cardiovascular effects of tacrine in the conscious dog: a role for catecholamines and vasopressin release

Centrally acting cholinergic agents are currently reported to increase blood pressure in various species through the stimulation of muscarinic cholinoceptors. Moreover, several cardiovascular adverse effects have been reported from clinical studies. The aim of this study was to investigate the effects of tacrine, an acetylcholinesterase inhibitor which has been reported to have therapeutic potential in Alzheimer's disease, on blood pressure and two vasopressor systems (sympathetic and vasopressinergic) in Beagle dogs. Intravenous (i.v.) tacrine (2 mg kg(-1)) induced, in conscious and anesthetized dogs, an increase in systolic and diastolic blood pressure, accompanied by bradycardia. This increase was dose-dependent with a peak effect at 1.5 min following administration. Tacrine also induced an increase in noradrenaline, adrenaline and vasopressin plasma levels. Pretreatment with the muscarinic receptor antagonist, atropine (2 mg kg(-1), i.v.), abolished the pressor response to i.v. injection of tacrine while pretreatment with the peripheral muscarinic receptor antagonist, methylscopolamine (0.2 mg kg(-1), i.v.), did not alter the increase in blood pressure. Similarly, noradrenaline and adrenaline changes in plasma levels were not modified by methylscopolamine but were abolished by atropine pretreatment. A similar tendency although not significant was observed for vasopressin plasma levels. The present results demonstrate that in dogs, tacrine (2 mg kg(-1), i.v.) stimulates central muscarinic cholinoceptors to increase blood pressure through activation of the two components of the sympathetic nervous system (i.e., neuroneuronal noradrenergic and the neurohormonal adrenergic pathways) as well as through increasing noradrenaline, adrenaline and vasopressin plasma levels.     

Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor

1. The role of nitric oxide (NO) in the regulation of acid secretion was examined in the anaesthetized rat. 2. A rat stomach was mounted in an ex vivo chamber, instilled with 2 ml of saline every 15 min, and the recovered sample was titrated at pH 7.0 against 0.1 N NaOH by use of an automatic titrator for acid secretion. Gastric mucosal blood flow (GMBF) was measured simultaneously by laser Doppler flowmeter. 3. Intragastric application of NO donors such as FK409 (3 and 6 mg ml[-1]) and sodium nitroprusside (SNP; 6 and 12 mg ml[-1]) as well as i.p. administration of cimetidine (60 mg kg[-1]), a histamine H2-receptor antagonist, significantly inhibited the increase in acid secretion in response to pentagastrin (60 microg kg(-1) h(-1), i.v.), in doses that increased gastric mucosal blood flow (GMBF). 4. Intragastric application of FK409 (6 mg ml[-1]) increased both basal and stimulated acid secretion induced by YM-14673 (0.3 mg kg(-1), i.v.), an analogue of thyrotropin-releasing hormone (TRH), but had no effect on the acid secretory response induced by histamine (4 mg kg(-1) h(-1), i.v.). 5. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg kg(-1), i.v.) did not affect basal acid secretion, but significantly potentiated the increase in acid secretion induced by YM-14673 and slightly augmented the acid secretory response to pentagastrin. 6. Both pentagastrin and YM-14673 increased the release of nitrite plus nitrate (NOx), stable NO metabolites, into the gastric lumen, and these changes were completely inhibited by prior administration of L-NAME (10 mg kg(-1), i.v.). 7. Pentagastrin caused an increase in luminal release of histamine and this response was significantly suppressed by intragastric application of FK409 (6 mg ml[-1]). 8. These results suggest that either exogenous or endogenous NO has an inhibitory action on gastric acid secretion through suppression of histamine release from enterochromaffin-like (ECL) cells.     

Effect of low dose aspirin on thrombus formation at arterial and venous microanastomoses and on the tissue microcirculation

In free flap/replantation surgery, failure is usually associated with thrombotic occlusion of a microvascular anastomosis (risk zone I) or, on occasion, flow impairment in the microcirculation of the transferred or replanted tissue (risk zone II). The objective of this study is to describe the effect of low dose aspirin on blood flow at both risk zones in microvascular surgery. Risk zone I: In rat femoral arteries and veins, thrombus formation was measured at the anastomoses using transillumination and videomicroscopy. Forty male Wistar rats were assigned in equal numbers to four groups: either arterial or venous injury with either aspirin (5 mg/kg systemically) or saline treatment. We found that aspirin significantly reduces thrombus formation at the venous anastomosis (p = 0.001). Risk zone II: In the isolated rat cremaster muscle downstream from an arterial anastomosis, we measured capillary perfusion, arteriolar diameters, and the appearance of platelet emboli for 6 hours in the muscle microcirculation. Sixteen male Wistar rats in two equal groups received either aspirin (5 mg/kg systemically) or saline. We found that in aspirin-treated animals, capillary perfusion is significantly (p = 0.002) improved, whereas arteriolar diameters and emboli only slightly increased. In conclusion, low dose aspirin inhibits anastomotic venous thrombosis and improves microcirculatory perfusion in our rat model. These studies provide quantitative data confirming and clarifying the beneficial effects of low dose aspirin in microvascular surgery.     

The effect of selective phosphodiesterase inhibitors on plasma insulin concentrations and insulin secretion in vitro in the rat

We have examined in rats the effects of Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[b]-thien-2-yl)-methyl-1-(2H)-p yridazinone), a selective inhibitor of type 3 phosphodiesterase (phosphodiesterase 3) and Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]-thiophene-2-carboximidamide HCl), an inhibitor of phosphodiesterase 3/4 on rat plasma insulin and glucose concentrations in pentobarbitone-anaesthetised rats and on insulin secretion by rat isolated islets. We have also compared their effects on islet phosphodiesterase activity. Org 9935 (0.1 and 1.0 mg kg(-1) i.v. 15 min previously) dose dependently elevated fasting and post-glucose (0.25 g kg(-1) i.v.) plasma insulin concentrations. Org 30029 in a dose of 10 mg kg(-1), but not 1 mg kg(-1), also increased plasma insulin concentrations. Neither drug modified either fasting or post-glucose plasma glucose concentrations. Each drug augmented glucose-induced insulin release by rat isolated islets in a static incubation system, with approximate EC50 values of 1.5 microM for Org 9935 and 20 microM for Org 30029. Phosphodiesterase activity, in both supernatant and pellet fractions of islet homogenates, was inhibited concentration dependently by each drug. Although the shape of the concentration-inhibition curve for Org 30029 precluded estimation of an IC50 value, this drug was clearly much less potent than Org 9935 (IC50 about 50 nM) in inhibiting islet phosphodiesterase activity. We conclude that the increase in plasma insulin produced by each drug is a consequence of augmented insulin secretion, probably secondary to inhibition of phosphodiesterase 3 in the islet beta cell, with a resultant elevation in cAMP. The failure of the drugs to modify plasma glucose may be due to concomitant inhibition of cAMP phosphodiesterase in liver and adipose tissue.