Endothelin-mediated vascular tone following focal cerebral ischaemia in the cat.

The actions of Bosentan and PD155080, nonpeptide 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 suprsylvian and ectosylvian gyri displayed marked dilatations, which were maintained in a population of vessel but differentiated into sustained constrictions in others. Perivascular subarachnoid microinjections of Bosentan (30 microM), PD155080 (30 microM), and artificial CSF (pH 7.2) were performed between 30 and 210 min following MCA occlusion. The perivascular microapplication of Bosentan (30 microM) and PD155080 (30 microM) around pial vessels overlying the suprasylvian and ectosylvian gyri, which are within the territory of the occluded MCA, elicited in increase in the calibre of postocclusion dilated and constricted pial arterioles. The perivascular microapplication of PD155080 (30 microM) 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 microM) 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 microM) 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 microM) 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.

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.

The effects of bosentan on cerebral blood flow and histopathology following middle cerebral artery occlusion in the rat.

The involvement of endothelins in the cerebrovascular events which follow a focal ischemic insult in the rat was explored in the present study. Intravenous (i.v.) administration of bosentan (3, 15 and 30 mg/kg), an endothelin ETA and ETB receptor antagonist, prior to middle cerebral artery occlusion in the rat did not significantly alter cortical perfusion in these rats. A 62 +/- 3% reduction in laser doppler flow was observed 10 min after middle cerebral artery occlusion in the vehicle-treated group compared to a 49 +/- 5% reduction in laser doppler flow in the group receiving 15 mg/kg bosentan. Pre-treatment with intravenous bosentan (15 mg/kg) prior to middle cerebral artery occlusion in the rat also failed to elicit significant alterations in the reduction in regional cerebral blood flow (frontal cortex; 81 +/- 13 ml/100 g/min) and subsequent hemispheric volume of ischemic damage observed (94 +/- 9 mm3) compared to the vehicle treated animals (68 +/- 9 ml/100 g/min, 113 +/- 5 mm3, respectively). Minimal changes were also observed in these endpoints, when a 15 mg/kg dose of bosentan was administered following middle cerebral artery occlusion. In conclusion bosentan failed to expose a major role for endothelins in focal ischemic pathology in the rat.

Rodent models of focal ischemia.

Experimental models of focal ischemia have provided an unparalleled insight into the dynamic events that surround ischemic brain injury. The dichotomous capacity of existing rodent models of focal ischemia to provide a controlled environment to examine the pathogenesis of focal ischemia and to allow assessment of the efficacy of potential therapeutic intervention is reviewed. The established rodent model of permanent middle cerebral artery occlusion (electrocoagulation) is critically examined and set against the distinctive features of novel methods that have been developed to reduce invasive surgery and to examine the pathological consequences of reperfusing a previously ischemic area. Emphasis has been placed on the technical requirements of each model that affect outcome and reproducibility.

Therapeutic potential of endothelin receptor antagonists in experimental stroke.

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 microM), 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 pre-injection baseline). Cerebral blood flow (measured by laser Doppler flowmetry) was reduced after MCA occlusion to 50% of preocclusion levels. Intravenous administration of PD156707 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.

Multiple origins of colonial green flagellates from unicells: evidence from molecular and organismal characters.

Phylogenetic hypotheses generated from cladistic analysis of organismal and molecular data are shown to be generally congruent and/or complementary for comparisons of unicellular and colonial green algae in the Chlorophyceae. Cladistic analysis of organismal character data corroborates the alliance of colonial Stephanosphaera with unicellular Haematococcus (Haematococcaceae sensu Smith), inferred from previous studies of nuclear-encoded rRNA sequence data. The organismal data also support monophyly of the colonial Volvocaceae (sensu Smith). Alliances of other unicellular taxa, including those ascribed to the "Euchlamydomonas" Hauptgruppe (sensu Ettl), are not resolved by organismal characters principally because the structure of the data is skewed to shared ancestral characters (symplesiomorphies) and unique characters (autapomorphies) which define individual taxa only. Reanalysis of rRNA sequence data, with additional sequence data for critical taxa, does not support monophyly of the colonial Volvocaceae (sensu Smith). However, these data are weak in the support of the alternate hypothesis of nonmonophyly. In contrast, relationships among most unicellular flagellates are unambiguously resolved by the molecular data. Although the failure of the sequence data to resolve relationships among colonial flagellates appears to be due to a sampling of conservative sequences, an ancient, rapid radiation event or taxon sampling bias may also be contributing to the ambiguity problem. Results from analysis of a combined data set (organismal and molecular) are generally consistent with the inferences of the organismal character data regarding the colonial flagellates and are also consistent with the inferences of the sequence data regarding the unicellular taxa.

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.

Possible location and function of neuropeptide Y receptor subtypes in the rat mesenteric arterial bed.

Earlier investigation of the vascular actions of Neuropeptide Y (NPY) led us to propose that distinct receptors mediated the prejunctional inhibition of periarterial nerve-stimulated norepinephrine (NE) release and the postjunctional potentiation of the increase in perfusion pressure elicited by vasoconstrictors. These receptors were designated Y2 and Y1, respectively, based on the ability of C-terminal fragments to mimic the former action. The present study investigates further the involvement of these putative receptor subtypes in the isolated and perfused mesenteric arterial bed. [Leu31Pro34]NPY, a novel analog with specificity at the Y1 receptors, potentiated the increase in perfusion pressure elicited by exogenously administered NE and arginine vasopressin, confirming the existence of this NPY subtype postjunctionally. This immediate and prolonged potentiation was abolished by phentolamine, attenuated by benextramine and the reputed NPY antagonist, PYX1. [11-36]NPY also produced a concentration-dependent potentiation of NE-stimulated increase in perfusion pressure suggesting that the Y2 receptor subtype may also be present postjunctionally in this model of the vascular neuroeffector junction. The finding that the profile of this potentiation differed from that elicited by [Leu31Pro34]NPY and, in contrast to the latter, was not attenuated by PYX1, intimates the existence of both distinct subtypes postjunctionally. [Leu31Pro34]NPY also reduced periarterial nerve-stimulated release of NE with a concomitant reduction in perfusion pressure indicating, in addition to the Y2 subtype, the presence of the Y1 receptor prejunctionally in the rat mesenteric arterial bed.

Central cardiovascular effects of rilmenidine and neuropeptide Y in the conscious spontaneously hypertensive rat: haemodynamic and biochemical evidence for a negative interaction.

The possible cardiovascular and biochemical interactions between the imidazoline ligand rilmenidine, a novel antihypertensive agent, and neuropeptide Y (NPY) were examined in spontaneously hypertensive rats (SHR). Rilmenidine (25-225 micrograms/kg) and NPY (1.7-15 micrograms/kg) both produced a significant, dose-dependent reduction in blood pressure (BP) after intracisternal (i.c.) administration in conscious SHR. When submaximal doses of rilmenidine (25 micrograms/kg) and NPY (1.7 micrograms/kg) were coadministered i.c., the resultant hypotension and bradycardia was less than either individual response and significantly less than the sum of their individual responses, suggesting the existence of an inhibitory interaction between these agents. To determine whether this interaction was evident at the second-messenger level, the effect of these agents on cyclic AMP levels was investigated in slices from the medulla oblongata of SHR. NPY (10(-6) and 10(-7) M) significantly inhibited forskolin-stimulated cyclic AMP production. Rilmenidine (10(-8)-10(-5) M) itself had no significant effect on forskolin-stimulated cyclic AMP levels in this system, but rilmenidine (10(-6) M) attenuated the inhibitory effect of NPY (10(-6) M) on cyclic AMP production. Thus, an inhibitory interaction between rilmenidine and NPY was observed at the hemodynamic and second-messenger level in the SHR.

Effects of neuropeptide Y on forskolin, alpha 2- and beta-adrenoceptor-regulated cAMP levels in the rat brain slice.

Neuropeptide Y (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels in slices of the medulla oblongata from WKY rats. No effect of NPY was observed on basal levels of cAMP in this region. Pretreatment with pertussis toxin (2 micrograms and 5 micrograms) IC prevented the reduction of forskolin-stimulated cAMP levels elicited by NPY in the medulla oblongata, suggesting that NPY is acting through an inhibitory guanine nucleotide binding protein to reduce cAMP accumulation. Moxonidine, an alpha 2-adrenoceptor agonist, was observed to reduce forskolin-stimulated cAMP levels in medullary slices. This inhibitory response was attenuated in the presence of NPY (10(-6) M). The beta-adrenoceptor agonist isoprenaline also elevated cAMP levels in the medulla oblongata; however, NPY did not alter this response. It is therefore proposed that the previously reported hemodynamic actions of NPY in the medulla oblongata, an area of cardiovascular significance, may be mediated via a reduction in cAMP levels. Moreover, an interaction between NPY and alpha 2-adrenoceptors, but not beta-adrenoceptors, on cAMP production in the medulla slice preparation was evident.

Endothelin-1-induced hypertension: a consequence of medullary ischemia?

The central hemodynamic effects of the peptide endothelin-1 (ET-1) have been investigated in the conscious, normotensive rat. Intracisternal administration of ET-1 (0.01-0.03 nmol) gave rise to an increase in mean arterial pressure with minimal effects on heart rate and was accompanied in some cases by barrel rolling activity. Intracisternal administration of 0.03 nmol ET-1 gave rise to a significant elevation in plasma noradrenaline and adrenaline levels. This elevation in plasma catecholamines was present only in those animals that also exhibited marked behavioral changes. Autoradiographic measurement of cerebral blood flow carried out during the maximum response to 0.03 nmol of intracisternal ET-1 revealed a widespread and profound ischemia throughout the caudal brainstem. Cerebral ischemia is known to activate compensatory circulatory reflexes in the medulla oblongata that result in increased sympathetic and vagal outflow. This is the most likely cause of intracisternal ET-1-induced hypertension. ET-1 is unique in its ability to override the brain's autoregulatory mechanisms and induce ischemia of pathological magnitude.

The cardiovascular actions of clonidine and neuropeptide-Y in the ventrolateral medulla of the rat.

1. The cardiovascular responses to neuropeptide-Y (NPY) (25 and 50 pmol) and clonidine (10 and 20 nmol) were examined following microinjection into the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM). Mean arterial pressure (MAP) and heart rate (HR) were measured in anaesthetized rats, pre- and post-injection. 2. The alpha 2-adrenoceptor agonist clonidine (10 and 20 nmol) reduced MAP and HR significantly when microinjected into the CVLM and RVLM. 3. NPY (25 and 50 pmol) microinjected into the CVLM decreased MAP and HR. However, in the RVLM neither dose had a significant cardiovascular effect. 4. The possibility of a functional interaction between the adrenergic system and NPY was examined by co-administration of clonidine and NPY in doses that gave submaximal blood pressure responses. In the CVLM this produced hypotension and bradycardia which was similar in magnitude to the sum of their individual responses, indicating that in this area their actions appear to be independent. 5. In the RVLM, where NPY has no significant cardiovascular effects, co-administration with clonidine, did not alter the response to clonidine. 6. It appears that in the areas investigated, there is no functional interaction between NPY and clonidine.