Toshal R. Patel

Endothelin-Mediated Vascular Tone Following Focal Cerebral Ischaemia in the Cat

The actions of Bosentan and PD155080, non-peptide endothelin receptor antagonists, were examined in feline pial arterioles in situ following middle cerebral artery (MCA) occlusion to gain insight into the cerebrovascular influence of endogenous endothelins in focal cerebral ischaemia. Immediately following permanent MCA occlusion, all pial arterioles overlying the suprasylvian and ectosylvian gyri displayed marked dilatations, which were maintained in a population of vessels but differentiated into sustained constrictions in others. Perivascular subarachnoid microinjections of Bosentan (30 μM), PD155080 (30 μM), and artificial CSF (pH 7.2) were performed between 30 and 210 min following MCA occlusion. The perivascular microapplication of Bosentan (30 μM) and PD155080 (30 μM) around pial vessels overlying the suprasylvian and ectosylvian gyri, which are within the territory of the occluded MCA, elicited an increase in the calibre of postocclusion dilated and constricted pial arterioles. The perivascular microapplication of PD155080 (30 μ/M) around postocclusion constricted arterioles overlying the ectosylvian and suprasylvian gyri elicited an increase in the calibre of arterioles (69 ± 49% from preinjection baseline; n = 8). The perivascular microapplication of Bosentan (30 μM) around postocclusion constricted arterioles overlying the ectosylvian and suprasylvian gyri also elicited an increase in the calibre of arterioles (68 ± 60% from preinjection baseline; n = 13). In contrast, the microapplication of CSF (pH 7.2) elicited small reductions in pial arteriolar calibre of postocclusion constricted arterioles (—8 ± 13% from preinjection baseline; n = 8). The perivascular microapplication of PD155080 (30 μM) around postocclusion dilated pial arterioles overlying the ectosylvian and suprasylvian gyri elicited an increase in the calibre of arterioles (11 ± 10% from preinjection baseline; n = 38). The perivascular microapplication of Bosentan (30 μM) around postocclusion dilated arterioles elicited an increase in the calibre of arterioles (16 ± 15% from preinjection baseline; n = 36). In contrast, the microapplication of CSF (pH 7.2) elicited small reductions in pial arteriolar calibre of postocclusion dilated arterioles (—9 ± 6% from preinjection baseline; n = 44). Perivascular microapplication of Bosentan or PD155080 had minimal effect on the calibre of pial arterioles on the parasagittal gyrus (anterior cerebral artery territory), although these arterioles had also displayed sustained dilatation following MCA occlusion. These results indicate that contractile factors (whose effects can be reversed with endothelin receptor antagonists) constrict or impair dilatation of cortical resistance arterioles in an acute cerebral ischaemic episode.

Therapeutic Potential of Endothelin Receptor Antagonists in Experimental Stroke

Summary: This investigation demonstrates an increase in endothelin (ET)-mediated vascular tone in peri-ischemic areas after experimental focal cerebral ischemia (middle cerebral artery occlusion) in the cat. Adventitial application of the butenolide antagonist PD155080 (30 μM), after MCA occlusions resulted in marked increases in caliber of dilated (10.6 ± 1.6% change from preinjection baseline) and constricted vessels (68.7 ± 17.5% change from preinjection baseline). Cerebral blood flow (measured by laser Doppler flowmetry) was reduced after MCA occlusion to 50% of preocclusion levels. Intravenous administration of PD 156707 30 min after MCA occlusion restored cerebral blood flow to preocclusion baseline levels at 6 h. The volume of ischemic damage in the cerebral hemisphere after MCA occlusion was significantly reduced (by 45%) after intravenous administration of PD156707.

Therapeutic Potential of Endothelin Receptor Antagonists in Cerebrovascular Disease

SummaryThe actions of the endothelins (endothelin-I. endothelin-2 and endothelin-3) are mediated via endothelin-A (EA) and endothelin-B (ETB) receptors. the former generally mediating vasoconstriction and the latter vasodilation.Peptide antagonists selective for either receptor sUbtype [e.g. BQ 123 (ETA) and BQ 788 (ETB)] and combined ETA/ETB receptor antagonists (e.g. PD 145065 and TAK 044) have been developed. More recently. small molecule non-peptide antagonists have also been synthesised. ETA receptor-selective agents include PD 155080 and BMS 182874. while Ro 46-2005 and bosentan are combined ETA/ETB receptor antagonists.The role of the endothelin family of vasoconstrictor peptides in the pathophysiology of cerebrovascular disease has been speculated upon. Increases in plasma and CSF levels of endothelin-I in delayed vasospasm following subarachnoid haemorrhage and acute ischaemic stroke have implicated the endothelins in these cerebrovascular diseases. The development of non-peptide endothelin receptor antagonists has facilitated investigations into the role of the endothelins in cerebrovascular disease.The endothelin receptor antagonists have been demonstrated to attenuate cerebral vasospasm following experimental subarachnoid haemorrhage in a variety of species. Additionally, the endothelin receptor antagonists ameliorate neuronal damage following eXIkrimental focal and global cerebral ischaemia. These actions have highlighted the therapeutic potential of endothelin receptor antagonists in cerebrovascular disease.

Effects on feline pial arterioles in situ of bosentan, a non-peptide endothelin receptor antagonist.

The cerebrovascular actions of bosentan, a novel endothelin antagonist with effects at endothelin ETA and ETB receptors, have been examined in individual pial arterioles on the cortical surface of chloralose-anaesthetised cats. Subarachnoid perivascular microapplication of bosentan (0.3-300 microM) had minimal effect on pial arteriolar calibre. Subarachnoid perivascular microapplication of endothelin (10 nM) effected a marked reduction in pial arteriolar calibre (reduced by 39.2 +/- 2.7% from baseline). This vasomotor effect of topical endothelin could be attenuated either by co-administration of bosentan (IC50 approximately 1 microM) or by the intravenous administration of bosentan (17 mumol/kg). These investigations suggest that bosentan (applied topically or systemically) may be a valuable tool in the elucidation of the functional significance of endothelins in the cerebral circulation in vivo.

Failure of an endothelin antagonist to modify hypoperfusion after transient global ischaemia in the rat.

The role of endogenous endothelins in mediating postischaemic hypoperfusion after transient global ischaemia was investigated in halothane-anaesthetised rats. Pretreatment with the broad-spectrum (ETA and ETB) endothelin antagonist, Bosentan (17μmol/kg) had minimal effect on postischaemic hypoperfusion, measured by hydrogen clearance, in the caudate nucleus and the parietal cortex in the 3 h after bilateral common carotid artery occlusion with concomitant haemorrhagic hypotension (transient global ischaemia). In a separate series of rats with CBF measured by [14C]iodoantipyrine autoradiography at 90 min after carotid occlusion with concomitant haemorrhagic hypotension, Bosentan treatment failed to significantly alter CBF in any of the 35 brain regions examined. No significant alterations in CBF, measured by hydrogen clearance, were observed after transient bilateral common carotid artery occlusion. [14C]Iodoantipyrine autoradiography at 90 min after occlusion failed to demonstrate any significant increases in CBF after transient bilateral common carotid artery occlusion in any of the 35 brain regions examined in anaesthetised rats. The failure of the broad-spectrum endothelin antagonist Bosentan, at concentrations known to inhibit the cerebrovascular effects of exogenous ET-1, provide no support for the view that endothelins have a major role in mediating acute postischaemic hypoperfusion.

The relationship between glutamate release and cerebral blood flow after focal cerebral ischaemia in the cat: effect of pretreatment with enadoline (a kappa receptor agonist).

The effect of the kappa-opioid agonist enadoline (CI-977) upon the relationship between cerebral blood flow and glutamate release was simultaneously assessed (using microdialysis and hydrogen clearance techniques respectively) at the same anatomical locus in the cerebral cortex (suprasylvian gyrus) after permanent middle cerebral artery (MCA) occlusion in halothane-anaesthetised cats. During controlled graded ischaemia, pretreatment with enadoline (0.3 mg/kg i.v. followed by continuous infusion at 0.15 mg/kg/h), initiated 30 min prior to MCA occlusion, significantly attenuated the marked increases in extracellular glutamate, aspartate and GABA observed in the focal ischaemic penumbra. The present data are consistent with the hypothesis that the neuroprotective efficacy of enadoline in focal cerebral ischaemia is due to inhibition of glutamate release in the ischaemic penumbra.

Endothelin receptor mediated constriction and dilatation in feline cerebral resistance arterioles in vivo

The receptors mediating the cerebrovascular actions of endothelins have been examined in feline cerebral resistance arterioles in vivo. The adventitial microapplication of the endothelin ETA receptor antagonist BQ-123 (cyclo D-aspartate-D-tryptophan-L-leucine-D-valine-L-proline) (0.1-10 microM) per se had minimal effect on cerebral resistance arterioles examined. The adventitial microapplication of endothelin-1 (10 nM) elicited a marked vasoconstriction of cerebral resistance arterioles (-29.1 +/- 1.9% from pre-injection baseline). The endothelin-1 induced vasoconstriction was attenuated, in a dose dependent manner, by the adventitial co-application of BQ-123 and endothelin-1 (estimated IC50 0.7 microM). The adventitial microapplication of the endothelin ETB receptor agonist BQ-3020 N-acetyl[Ala11,Ala15]ET-1 (6-21)) (0.001-1 microM) effected a dose dependent vasodilatation (EC50 30 nM, maximum response 25 +/- 5% from pre-injection baseline). The magnitude of the vasodilatation elicited by BQ-3020 (100 nM and 1 microM) was dependent on the pre-injection calibre of the arterioles examined. The intracarotid infusion (via the lingual artery) of BQ-3020 (0.5-500 pmol/min) had no significant effect on the calibre of cerebral resistance arterioles. These results suggest that the peptide endothelin ETB receptor agonist fails to gain access to the cerebrovascular endothelin ETB receptors following its intraluminal administration. These investigations indicate that endothelin ETA receptors mediate vasoconstriction and endothelin ETB receptors mediate vasodilatation in feline cerebral resistance arterioles in vivo.

AMPA receptor antagonism attenuates MK-801-induced hypermetabolism in the posterior cingulate cortex

The effect of pretreatment with an AMPA receptor antagonist, NBQX, on MK-801-induced alterations in glucose use was examined using [14C]-2-deoxyglucose autoradiography. NBQX (7 mg/kg) had minimal effect on glucose utilisation in all anatomical regions examined. The intravenous administration of MK-801 (0.2 mg/kg) induced increases in glucose use in the limbic system and cingulate cortex. MK-801 reduced glucose utilisation in the sensory motor and auditory cortices. Pretreatment with NBQX attenuated the MK-801-induced hypermetabolism in the posterior cingulate cortex. The decreases in glucose utilisation induced by MK-801 were not exacerbated by the pretreatment with NBQX. The interaction between NBQX and MK-801 suggests a possible method of attenuating some of the adverse effects of the non-competitive NMDA receptor antagonists in the posterior cingulate cortex.

Postischemic hypoperfusion in transient global ischemia : a role for endothelins ?

Summary: The role of endogenous endothelins (ETs) in mediating postischemic hypoperfusion after transient global ischemia was investigated in halothane-anesthe-tized rats. Pretreatment with the broad spectrum ETA and ETB antagonist bosentan (17 μmol/kg) had minimal effect on postischemic hypoperfusion, as measured by hydrogen clearance, in the caudate nucleus and the parietal cortex during 3 h after bilateral common carotid artery occlusion with concomitant hemorrhagic hypotension (transient global ischemia). In cerebral blood flow (CBF) measured by [14C]iodoantipyrine autoradiography at 90 min after carotid occlusion with concomitant hemorrhagic hypotension, bosentan treatment failed to alter CBF in any of the cerebral cortical regions examined. No changes in CBF, as measured by hydrogen clearance, were observed after transient bilateral common carotid artery occlusion. [14C]Iodoantipyrine autoradiography at 90 min post occlusion failed to demonstrate any increases in cerebral blood flow after transient bilateral common carotid artery occlusion in any of the 35 brain regions examined in anesthetized rats.

The Use of Microdialysis for Monitoring the Effect of the Neuroprotective Drug CI-977 on Extracellular Excitatory Amino Acids

The excitatory amino acid glutamate is now accepted as an important cause of brain damage in animals following ischemia [1, 2]. Its effects can be ameliorated by various neuroprotective drugs that either block the postsynaptic N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors or which putatively inhibit the presynaptic release of glutamate [3]. In man, ischemia is a cause of brain damage, not only in stroke but also in head injury; as many as 85% of patients who die following a head injury have evidence of hypoxic ischemic brain damage [4, 5]. The use of neuroprotective drugs in these conditions could be beneficial, and there are currently several clinical studies under way.