Inhibition of alpha4 integrin protects against transient focal cerebral ischemia in normotensive and hypertensive rats.

BACKGROUND AND PURPOSE: The present study was performed to determine the role of alpha4 (CD49d), a member of the integrin family of adhesion molecules, in ischemic brain pathology. METHODS: Male spontaneously hypertensive rats (SHR) or Sprague-Dawley rats underwent 60-minute middle cerebral artery occlusion (MCAO) followed by 23-hour reperfusion. Animals were injected intravenously with 2.5 mg/kg anti-rat alpha4 antibody (TA-2) or isotype control antibody (anti-human LFA-3 IgG(1), 1E6) 24 hours before MCAO. Infarct volume was quantified by staining of fresh tissue with tetrazolium chloride and myeloperoxidase activity measured in SHR tissue homogenates 24 hours after MCAO. In SHR, mean arterial blood pressure was recorded before and after MCAO in animals treated with TA-2 and 1E6. Fluorescence-activated cell sorting analysis was performed on peripheral blood leukocytes before and after MCAO. RESULTS: TA-2 treatment significantly reduced total infarct volume by 57.7% in normotensive rats (1E6, 84.2+/-11.5 mm(3), n=17; TA-2, 35.7+/-5.9 mm(3), n=16) and 35.5% in hypertensive rats (1E6, 146.6+/-15.5 mm(3), n=15; TA-2, 94.4+/-25.8 mm(3), n=11). In both strains, TA-2 treatment significantly reduced body weight loss and attenuated the hyperthermic response to MCAO. In SHR, treatment with TA-2 significantly reduced brain myeloperoxidase activity. Resting mean arterial blood pressure was unaffected by treatment. Leukocyte counts were elevated in TA-2-treated rats. Fluorescence-activated cell sorting analysis demonstrated the ability of TA-2 to bind to CD3+, CD4+, CD8+, and CD11b+ cells in both naive animals and after MCAO. CONCLUSIONS: These data demonstrate that inhibition of alpha4 integrin can protect the brain against ischemic brain injury and implicate endogenous alpha4 integrin in the pathogenesis of acute brain injury. The mechanism by which alpha4 integrin inhibition offers cerebroprotection is independent of blood pressure modulation and is likely due to inhibition of leukocyte function.

Angiotensin-converting enzyme, bradykinin, angiotensin, and cerebral vessel reactivity.

Bradykinin (BK) produced concentration-related relaxations of cat middle cerebral arteries and was ineffective in cat basilar arteries. On rabbit basilar arteries, BK initially produced concentration-related relaxations; however, when repeated at 2-hour intervals, BK eventually produced pure contractile responses. After preincubation of the tissues with cycloheximide, BK produced reproducible relaxation responses. The angiotensin-converting enzyme inhibitors, SQ 14,225, BPP5a, and BPP9a, had no effect on the concentration-effect curves of BK, AII, or 5-HT with any of the preparations, but responses to AI were inhibited. These results suggest that, in these tissues, angiotensin-converting enzyme is important for conversion of AI to AII, but apparently not for the degradation of BK.

Design, synthesis, and in vitro activity of bis(succinimido)hexane peptide heterodimers with combined B1 and B2 antagonist activity.

We have developed a series of peptide heterodimers based on the B2 antagonist D-Arg0-[Hyp3,D-Phe7,Leu8]-BK (1) and the B1 antagonist Lys0-[Leu8,des-Arg9]-BK (7) that are potent antagonists of both B1 and B2 receptors. From this series, compound 50 (alternatively, CP-0364), the 1,6-bis(succinimido)hexane heterodimer of D-Arg0-[Hyp3,Cys6,D-Phe7,Leu8]-BK (2), and D-Arg0-[Cys1,Hyp3,Leu8,des-Arg9]-BK (6), was found to be the most active both in vitro and in vivo. Compound 50 has a pA2 of 8.3 when measured against bradykinin (BK)-induced rat uterine smooth muscle contraction and an IC50 of approximately 10(-8) M against [des-Arg9]-BK-induced rabbit aorta smooth muscle contraction in vitro. Compounds such as 50 may be useful in the treatment of both subacute and chronic inflammatory disorders wherein both B2 and B1 receptors appear to contribute to the clinical manifestations of the disease.

Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

A new class of bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers.

A systematic study on the dimerization of the bradykinin (BK) antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Leu8-Arg9 has been performed. The first part of this study involved compounds wherein dimerization was carried out by sequentially replacing each amino acid with cysteine and cross-linking with bismaleimidohexane. The second part of this study utilized a series of bissuccinimidoalkane dimers wherein the intervening methylene chain was varied systematically from n = 2 to n = 12 while the point of dimerization was held constant at position 6. The biological activities of these dimers were then evaluated on BK-induced smooth muscle contraction in two different isolated tissue preparations: guinea pig ileum (GPI) and rat uterus (RU). Several of the dimeric BK antagonists displayed remarkable activities and long durations of action. In addition, dimerization at position 4, 7, 8, or 9 produced dimeric analogues with markedly reduced potency. Rank order of antagonist potency as a function of dimerization position is as follows: rat uterus, 6 greater than 5 greater than 0 greater than 2 greater than 1 greater than 3 much greater than 4, 7, 8, 9; guinea pig ileum, 6 greater than 5 greater than 3 greater than 2 greater than 1 greater than 0 much greater than 4, 7, 8, 9. Evaluation of the linker length as represented by the number of methylene units indicated an optimal distance between the two monomeric peptides of six to eight methylene moieties. These studies also revealed that the carbon-chain length significantly affected the duration of action in vitro and resulted in partial agonism effects when n greater than 8. The optimum activity in vitro was achieved with dimerization at position 6 and n = 6 (designated herein as compound 25; alternatively, CP-0127). Similar effects in potency were also seen when the monomeric antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Phe8-Arg9 (NPC-567) was dimerized using similar chemistry. These results suggest that the development of BK antagonists of significant therapeutic potential may be possible using a dimerization strategy that can overcome the heretofore limiting problems of potency and in vivo duration of action found with many of the BK antagonists in the literature.

The action of bradykinin and oxytocin on the isolated whole uterus and myometrium of the rat in oestrus.

1. A technique is described for obtaining a myometrial preparation devoid of endometrium, from the uterus of the rat in oestrus. 2. Acetylcholine and prostaglandin F2alpha (PGF2alpha) produced concentration-effect curves with the same maximal tensions and slope on the whole uterus and myometrial preparations. Concentration-effect curves to bradykinin and oxytocin on the myometrial preparation were altered, resulting in a shift to the right and a decreased maximum response compared with those produced by the whole uterus. 3. Indomethacin produced greater antagonism of the responses of the whole uterus to bradykinin and oxytocin than to acetylcholine and PGF2alpha, whereas responses of the myometrium to all four agonists were similarly depressed. 4. Responses of the myometrial preparation to a range of concentrations of bradykinin and oxytocin were significantly enhanced by prior sensitization of the myometrium to PGF2alpha. This significant enhancing effect of PGF2alpha was only seen with the threshold dose of acetylcholine. 5. It appears that the mechanism of action of bradykinin and oxytocin on the rat uterus involves both a direct action and an indirect action. The indirect action possibly involves release of prostaglandin(s) from the endometrium.

Effect of oestrogens and progesterone alone and in combination on the female rat plasma kininogen concentrations.

PIP: The effect of estrogens and progesterone alone and in combination on virgin, female rat plasma kininogen levels was studied. Doses of 5 and 10 mcg/kg/day beta-estradiol and 50 and 250 mcg 2,4dimethyl-5,5-diphenyl pent-4-enoic acid sodium salt for 5 days gave a dose response effect. Doses of .5, 2.5, and 5 mg/kg progesterone had no effect on kinin precursor concentration. However, when varying doses of progesterone were used in conjunction with estrogen, a marked increase in kininogen concentrations produced by the estrogens was reduced by, and in proportion to, the dose of progesterone. The effect was more pronounced with the lower doses of estrogen. It appeared that progesterone reduced the increase in kinin precursor produced by the estrogen alone, in proportion to the estrogen/progesterone ration.^ieng

Beta-adrenoceptor agonist activity of labetolol on the isolated uterus of the rat.

Isoprenaline produced dose-dependent reductions of responses of the isolated uterus of the rat produced by an EC(80) of acetylcholine. Propranolol acted as a competitive antagonist to isoprenaline. Labetolol also reduced the acetylcholine-induced contractions but was much less potent than isoprenaline. The greatest reduction was smaller than that produced by isoprenaline. Propranolol antagonized the lower doses of labetolol. It is suggested that labetolol possesses partial agonist activity at the beta-adrenoceptors of the rat isolated uterus.

Changes in plasma kininogen levels induced by cellulose sulphate during pregnancy in the rat.

1 Cellulose sulphate (1 mg/kg) produced a 30-40% depletion of plasma kininogen in rats.2 The time course of repletion of kininogen in the plasma was compared in rats in the oestrous and dioestrous stages of the cycle and in 22 day pregnant animals. A partial repletion occurred, 3 h after the cellulose sulphate injection, which was followed by a secondary fall in plasma kininogen. Plasma kininogen values were back to control levels 10 h after the treatment in all groups.3 Treatment of rats from days 19-22 of pregnancy with cellulose sulphate resulted in 40% depletion of plasma kininogen and in prolongation of pre-parturition behaviour.4 It is suggested that the increase which normally occurs in plasma kininogen levels towards the end of pregnancy in the rat may play a role in the process of parturition.

Attenuation of arterial blood pressure fall in endotoxin shock in the rat using the competitive bradykinin antagonist Lys-Lys-[Hyp2, Thi5,8, DPhe7]-Bk (B4148).

The selective competitive bradykinin (Bk) antagonist, B4148 (Lys-Lys-[Hyp2, Thi5,8, DPhe7]-Bk) infused at 100 micrograms kg-1 min-1 into rats produced a significant inhibition of the hypotensive effect of Bk and had no effect against acetylcholine-induced responses. In a rat model of endotoxin shock, the fall in mean arterial blood pressure in response to an intravenous injection of lipopolysaccharide from E. coli was significantly attenuated by the same infusion of B4148 compared to controls. These findings suggest that kinins are involved in the hypotensive response to endotoxin shock in rats. The development of potent Bk antagonists offers a new experimental approach for evaluating the role of kinins in this and other disease states and potential therapy in such disorders.

5-HT1-like receptors mediate 5-hydroxytryptamine-induced contraction of human isolated basilar artery.

1. The 5-hydroxytryptamine (5-HT) receptor mediating contraction of endothelium denuded human basilar artery has been characterized in vitro. 2. 5-HT and a variety of 5-HT agonists contracted human isolated basilar artery with a rank order of agonist potency, 5-carboxamidotryptamine (5-CT) greater than 5-HT identical to methysergide greater than GR43175 much greater than 8-OHDPAT much greater than 2-methyl-5-HT. The maximum response produced by these agonists differed. 3. None of the agonists relaxed human basilar artery when tone was elevated with prostaglandin F2 alpha, indeed further contraction was seen. 4. The contractile responses of human basilar artery to 5-HT and the selective 5-HT1-like agonist GR43175 were highly reproducible whilst those to 5-CT were not. 5. The contractile response to both 5-HT and GR43175 was resistant to antagonism by ketanserin and GR38032, thus excluding activation of 5-HT2 and 5-HT3 receptors. The contractile action of 5-HT and GR43175 was also not antagonized by (+/-)-cyanopindolol, excluding the activation of receptors similar to 5-HT1A and 5-HT1B recognition sites identified in ligand binding studies. 6. In marked contrast, methiothepin was a potent antagonist of the contractile actions of both 5-HT and GR43175, with a pA2 value of 8.8 against both agonists. Methiothepin (100 nM) had no effect on the contractile response to the thromboxane A2-mimetic U46619. 7. We conclude that 5-HT and GR43175 contract the human isolated basilar artery by activating the same receptor type. This receptor appears identical to the 5-HT1-like receptor causing contraction of the dog isolated saphenous vein and cerebral blood vessels from the dog and primate.

New synthetic antagonists of bradykinin.

1 A new synthetic bradykinin analogue was found to be an antagonist of bradykinin-induced vascular permeability in rabbit skin. It was effective in equimolar concentrations. 2 These analogues also antagonized the action of bradykinin in contracting the guinea-pig isolated ileum. The mean pA2 values of five different antagonists ranged from 5.3-6.4 respectively, on this preparation. 3 Our observations, together with those of others suggest that these antagonists act on the same receptor types, viz., B2, in rabbit blood vessels and in smooth muscle of guinea-pig ileum. 4 Our results support the view that the way is now promising for the synthesis of potent specific antagonists of bradykinin for experimental and therapeutic use.

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Bradykinin B2, but not B1, receptor antagonism has a neuroprotective effect after brain injury.

The aim of the present study was to measure the therapeutic effects of bradykinin antagonists on lesion volume and brain swelling induced by cold injury in the parietal cortex of rat and mouse, respectively. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (3 mm diameter) to the intact dura of rat and mouse for 6 and 30 sec, respectively. At 24 h after the injury, the brains were removed and lesion volume was determined by the triphenyltetrazolium chloride method in rats. In the mouse, brain swelling was expressed as percentage increase in weight of the injured hemisphere which is compared to the contralateral side. After a subcutaneous priming dose of 18 microg/kg, a 1-h pretreatment and 24-h posttreatment using osmotic minipumps (300 ng/kg x min) was applied. Hoe140, a bradykinin receptor 2 antagonist, revealed a 19% reduction of lesion volume (p < 0.05) in the rat and a 14% diminution of brain swelling (p < 0.05) in the mouse. In contrast, the bradykinin receptor 1 antagonist, B 9858, had no effect on lesion volume compared to sham treated rats. When B 9858 was given in combination with Hoe140, a significant reduction in lesion volume was seen which was equivalent to and not different from that seen with Hoe140 alone in the rat. We conclude that brain injury after cold lesion is partially mediated by bradykinin and can be successfully treated with B2 antagonists.

Serotonin and migraine.

Migraine has long been considered as a "vascular headache" but clearly neurological mechanisms are involved. The pathophysiology appears to somehow involve serotonin, both peripherally and centrally, but its involvement may be just epiphenomenal. Adding to the enigma it is apparent that many of the presently available drugs for the treatment of migraine interact in one way or another with serotonin receptors. However, they tend to have a number of other unrelated actions and they are only of limited clinical value. Interestingly a promising new drug for the treatment of the acute attack, sumatriptan, has a very selective action as an agonist at a specific 5-HT1-like receptor sub-type, mediating vasoconstriction, which is localized on cranial blood vessels. Its action may, or may not, be independent of any involvement of serotonin in the genesis of migraine. Hopefully though, current attempts to determine sumatriptan's mechanism of action will shed further light on the pathology of migraine itself and the putative involvement of serotonin.

Cerebrovascular reactivity to angiotensin and angiotensin-converting enzyme activity in cerebrospinal fluid.

The purpose of the present study was to test the vasomotor effect of angiotensin I (A I) and angiotensin II (A II) in feline cerebral arteries and to examine the presence of angiotensin converting enzyme (ACE) activity in the vessel wall and cerebrospinal fluid (CSF). A II (10(-8) -10(-5) M) induced concentration-dependent contractions of feline pial arteries (resting diameter, 98-286 microns) in situ with a maximum of 34% at 10(-4) M A II. A I produced dose-related contractions being approximately 20 times less potent than A II. The action of A I was significantly attenuated by the ACE inhibitor captopril (10(-5) M). These findings demonstrate the presence of ACE activity in the vessel wall and/or its surroundings. ACE activity was also found in feline CSF sampled from the cisterna cerebellomedullaris. Bradykinin (BK) was broken down and A I converted to A II by CSF, both effects being inhibited by captopril. This was demonstrated using bioassay and high-performance liquid chromatography. Considering the present and previous studies we conclude that the presence of ACE in the vessel wall and CSF is necessary for the conversion of A I to A II. Although ACE in CSF is able to degrade BK it appears not to be important for the metabolism of BK acting from the perivascular side of pial arteries in situ.

Rationale for the use of 5-HT1-like agonists in the treatment of migraine.

Migraine headache is thought to be associated with a dilatation of cranial blood vessels, particularly those in the dura mater, and an accompanying localized sterile inflammatory response. Sumatriptan is a highly selective 5-HT1-like receptor agonist which selectively constricts cranial blood vessels (including those in the dura mater). It also inhibits neurogenically-mediated plasma protein extravasation in the dura mater. Haemodynamic studies in anaesthetized animals have shown that sumatriptan selectively constricts the carotid arterial circulation and this effect appears to be restricted to an effect on carotid arteriovenous anastomoses. Sumatriptan has a much more selective pharmacological profile than ergot preparations which are also used in the acute treatment of migraine. The development of sumatriptan has been based on a vascular theory of migraine and its high degree of efficacy in the treatment of migraine strengthens the argument that dilatation of cranial blood vessels is the cause of vascular headache.

Angiotensin converting enzyme inhibitors and expression of des-Arg9-BK (kinin B1) receptors in vivo.

The effect of the selective kinin B1 receptor agonist des-Arg9-BK was studied on blood pressure and on the in vitro aorta of rabbits pretreated 18 h earlier with lipopolysaccharide from E. coli, an infusion of bradykinin or with one of three angiotensin converting enzyme inhibitors captopril, enalapril or teprotide. The hypotensive response in vivo and contractile response seen on the in vitro aorta was selectively increased to des-Arg9-BK in all pretreated groups compared to controls, effects which were blocked by the selective competitive kinin B1 receptor antagonist des-Arg9-[Leu8]BK. Dexamethasone given to lipopolysaccharide pretreated rabbits had no effect on the increased hypotensive response seen with des-Arg9-BK. The skin vascular permeability response to des-Arg9-BK, bradykinin and histamine remained unchanged in the groups pretreated with lipopolysaccharide or captopril compared to controls. The possible mechanism(s) whereby angiotensin converting enzyme inhibitors produce this effect and the possible relevance to the inflammatory side-effects seen with this group of drugs is discussed.

Evidence for the presence of 5-HT1-like receptors in rabbit isolated basilar arteries.

The 5-hydroxytryptamine (5-HT) receptor mediating contraction of rabbit isolated endothelium denuded basilar artery has been investigated. 5-HT and a variety of 5-HT receptor agonists contracted rabbit isolated basilar artery with a rank order of agonist potency: 5-carboxamidotryptamine (5-CT) greater than 5-HT greater than GR43175. None of these agonists relaxed rabbit isolated basilar artery when tone was elevated with prostaglandin F2alpha. The contractile response to both 5-HT and GR43175 was resistant to antagonism by GR38032, phentolamine, (+/-)-cyanopindolol and yohimbine. Ketanserin (100 nM) and mesulergine (100 nM) produced small significant rightward shifts of C-E curves to 5-HT with respective concentration-ratio shifts of 5.7 (1.5-21.0 95% confidence interval and 2.89 (1.1-7.6 95% confidence interval). GR43175-induced contraction was resistant to antagonism by ketanserin however the maximum response to GR43175 was significantly reduced in the presence of mesulergine, with no change in EC50. Methiothepin was a potent antagonist of the contractile actions of both 5-HT and GR43175, with respective pA2 values against each agonist of 10.3 and 9.9. The slope of the Schild regression for methiothepin against 5-HT-induced contraction was significantly less than unity. Methiothepin (100 nM) had no effect on the contractile response to the thromboxane A2 mimetic U-46619. It is concluded that 5-HT and GR43175 contract rabbit isolated basilar artery by activating a 5-HT1-like receptor. In addition 5-HT may activate a population of 5-HT2 receptors producing a further contraction of rabbit isolated basilar artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile effects of 8-hydroxy-2-(di-n-propylamino)tetralin and flesinoxan in human isolated basilar artery.

The aim of the present study was to assess the effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and flesinoxan in ring preparations of human basilar artery. 5-Hydroxytryptamine-(5-HT), 8-OH-DPAT and flesinoxan induced concentration-dependent contractions of human basilar artery, the rank order of agonist potency being 5-HT greater than 8-OH-DPAT approximately flesinoxan. The rank order of maximum response, relative to 5-HT was 5-HT (100%) much greater than 8-OH-DPAT (40.4 +/- 4.4%) much greater than flesinoxan (7.0 +/- 2.3%). The contractile effects of 8-OH-DPAT were blocked by phentolamine (10 microM) but not by labetalol (10 microM). Spiperone (1 microM) had no significant effect on either 5-HT or 8-OH-DPAT-induced contraction, however methiothepin (100 nM) produced inhibition of both 5-HT- and 8-OH-DPAT-induced contraction of human basilar artery. In addition, flesinoxan (100 microM) produced blockade of 5-HT-, 8-OH-DPAT- and sumatriptan (a 5-HT1-like receptor agonist)-induced contraction of human basilar artery, although full concentration-effect curves were not obtained. In some preparations 8-OH-DPAT produced a concentration-dependent relaxation of tone. This effect was particularly apparent in the presence of phentolamine. We conclude from the relative rank order of antagonist potency that 8-OH-DPAT and 5-HT produce contraction of the human basilar artery by activation of the same receptor, a 5-HT1-like receptor distinct from the 5-HT1A receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)