Effects of adenosine and its analogues on porcine basilar arteries: are only A2 receptors involved?

The aim of this study was to test the effect of adenosine and four of its analogues, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CADO), L-phenylisopropyladenosine (L-PIA), and N6-cyclohexyl-adenosine (CHA), on prostaglandin (PG) F2 alpha-constricted pig basilar arteries, and from their rank order of potency determine the receptor type involved. The order of potency for the relaxation of the PGF2 alpha constriction was NECA greater than adenosine, 2-CADO greater than L-PIA greater than CHA, which is in keeping with the A2 receptor subtype. The study also investigated the effects of a known adenosine antagonist, namely, the xanthine derivative 8-phenyltheophylline, which at concentrations having no intrinsic effect (10(-8) and 10(-7) M) produced a significant shift to the right only for the NECA dose-response curve.     

Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide: with detailed description of the closed cranial window technique in rats

This study describes a closed cranial window technique that allows the observation and measurement of rat pial arterioles and venules in situ. The resolving power of this system is 1-2 microns. Using this sensitive technique, we characterized the responses to 7% carbon dioxide inhalation and adenosine in arterioles (10-70 microns) and venules (15-100 microns). During carbon dioxide inhalation, larger arterioles (greater than 40 microns) dilated more than smaller arterioles (less than 20 microns). There was limited vasoreactivity of pial venules during CO2 inhalation. Dilation of arterioles was initially observed with an adenosine concentration of 10(-8) M. Almost a twofold increase in diameter was noted at 10(-3) M. In contrast to the effect of CO2 inhalation, the degree of dilation with topical application of adenosine was not size dependent. Pial venules did not respond to adenosine. The technique for observation of pial vessels using the closed cranial window and for measurement of vessel diameter by video camera system microscopy is a powerful tool for studying in vivo the cerebral circulation in the rat.     

Cerebral circulation and histamine: 2. Responses of pial veins and arterioles to receptor agonists

H2-receptors predominantly mediate pial arteriolar dilatation in response to histamine, but the reaction of pial veins to histamine has not been clearly identified. In anesthetized cats, we examined responses of pial veins and arterioles to perivascular microapplication of histamine and specific histamine H1 and H2 receptor agonists. Arterioles were very sensitive to the H2-receptor agonist impromidine, with significant dilatation (+16%) occurring at concentrations as low as 10(-10) M. Arteriolar responses to H1 receptor stimulation by 2.2-pyridylethylamine were small, even at high concentrations. The order of potency and maximum dilatations found for the receptor agonists were: H2 (43%) greater than histamine (28%) greater than H1 (17%). By contrast, pial veins did not respond to histamine or the receptor agonists. The results indicate that pial venomotor activity to histamine is negligible, and suggest a sparse distribution of histamine receptors on the outer surfaces of pial veins.     

Effects of cocaine on pial arterioles in cats

We used the closed cranial window technique to observe the responses of pial arterioles to topical application of cocaine in 29 anesthetized cats. Alterations in arteriolar diameter were dependent on the concentration of cocaine applied. Cocaine dissolved in artificial cerebrospinal fluid at concentrations of 10(-8) or 10(-7) M was without effect. Concentrations of 10(-6) and 10(-5) M produced dilation (4.9 +/- 1.5% [mean +/- SEM] and 5.9 +/- 2.0%, respectively) in large arterioles (greater than 100 microns) but no significant change in the diameter of small arterioles (less than 100 microns). A concentration of 10(-4) M dilated both large and small arterioles (20.3 +/- 3.1% and 12.0 +/- 7.1%, respectively). Pretreatment with 1 mg/kg i.v. propranolol blocked the increase in pial arteriolar diameter after application of 10(-4) M cocaine and produced significant vasoconstriction in small arterioles (-8.3 +/- 3.1%). Cocaine produces vasodilation of cat cerebral arterioles. This effect appears to be mediated, at least in part, by mechanisms that depend on stimulation of beta-adrenergic receptors.     

Human retinal pigment epithelial cells in culture possess A2-adenosine receptors

Adenosine agonists cause a marked stimulation in cyclic AMP accumulation in whole human retinal pigment epithelial (RPE) cells in the presence of adenosine deaminase and papaverine, a phosphodiesterase inhibitor. N-Ethylcarboxamidoadenosine (NECA) stimulates cyclic AMP accumulation 16.1-fold above basal with an EC50 of 2.5 x 10(-7) M. It is also an effective (1.9-fold) stimulator of adenylate cyclase activity in RPE membrane preparations and a modest (1.22-fold) stimulator in the presence of forskolin in RPE cell membranes prepared from freshly isolated porcine RPE. N6-Cyclopentyladenosine (CPA) and N6-phenylisopropyladenosine (PIA) also increase cyclic AMP levels with EC50s of 4.9 x 10(6) M (8.9-fold above basal) and 3.5 x 10(-6) M (8.0-fold above basal) respectively. This potency order (NECA greater than PIA greater than CPA) is typical of A2-adenosine receptors. The relatively A1-selective agonists 10(-7) M indicating that RPE cells do not have A1-receptors which inhibit adenylate cyclase. Three adenosine receptor antagonists, BW-A1433U, 8-cyclopentyltheophylline and 8-sulfophenyltheophylline, blocked the NECA-induced stimulation of cyclic AMP accumulation with IC50s of 0.36 microM, 1.5 microM, and 75 microM respectively. Since alteration of cAMP levels has been demonstrated to affect several RPE functions, including cell migration, resorption of subretinal fluid, and phagocytosis, adenosine may play a significant regulatory role in RPE.     

Coronary vascular effects of vasoactive intestinal peptide in the isolated perfused rat heart

The potency and mechanism of action of vasoactive intestinal peptide (VIP) for producing coronary vasodilation was investigated in the isolated perfused heart of the rat. VIP reduced coronary vascular resistance in a dose-dependent manner, starting at 1 x 10(-10) M, and maximally reduced coronary vascular resistance by 49% at 1 x 10(-8) M. The potency of VIP for reducing coronary vascular resistance (EC50=3.02 x 10(-10) M) was considerably greater than that of adenosine (EC50=6.17 x 10(-8) M) and sodium nitroprusside (EC50=2.45 x 10(-6) M). The vasodilatory action of VIP was more easily observed after increasing vascular tone by perfusion of the hearts with a modified physiological solution containing reduced concentrations of potassium (3.2 mM) and calcium (1.2 mM). Under these conditions, VIP maximally reduced coronary resistance by 54% at 7 x 10(-9) M. The potency of VIP for reducing coronary resistance in these hearts, however, decreased 16-fold (EC50=4.90 x 10(-9) M) while that of SNP remained unaltered (EC50=3.39 x 10(-6) M), compared with hearts perfused with higher levels of potassium (5.9 mM) and calcium (2.5 mM). The vasodilatory effect of VIP occurred without a significant change in heart rate, myocardial contractility or oxygen consumption. In additional studies, the dose-dependent effect of VIP on cyclic nucleotide release from the heart was determined by infusing VIP into the coronary circulation in a cumulative fashion to produce final concentrations between 1 x 10(-11) and 1 x 10(-9) M. VIP increased cyclic AMP at 1 x 10(-9) M but did not increase cyclic GMP. Studies using RT-PCR and immunohistochemistry clearly demonstrated the presence of two VIP receptor subtypes, VPAC1 and VPAC2, in the arteries and arterioles of the heart. In conclusion, VIP is a potent vasodilator in the coronary circulation of the rat and the role of VIP in the control of coronary vascular resistance depends on the circulating levels of potassium and calcium. This vasodilatory effect involves binding to specific coronary cell surface receptors, VPAC1 and/or VPAC2, and is dependent on cyclic AMP only during maximal vasodilation.     

Selective attenuation by perivascular blood of prostanoid-dependent cerebrovascular dilation in piglets

Cerebral hemorrhagic insults are common in neonates. However, the consequences of intracranial blood on cerebral hemodynamics are poorly understood. We examined the effects of perivascular blood on cerebrovascular dilator responses in 29 piglets. Fresh, autologous blood (n = 15) or cerebrospinal fluid (n = 14) was placed under the dura mater over the parietal cortex, and the piglets were allowed to recover from anesthesia. One to four days later, a closed cranial window was placed over the parietal cortex and pial arteriolar responses to arterial hypercapnia (PaCO2 greater than 55 mm Hg), hemorrhagic hypotension (mean arterial blood pressure less than 35 mm Hg), or topical application of 10(-6) and 10(-4) M isoproterenol were determined. Pial arterioles in the cerebrospinal fluid group dilated 27 +/- 4% (mean +/- SEM) (n = 11) in response to hypercapnia, 26 +/- 5% (n = 9) in response to hypotension, and 26 +/- 3% in response to 10(-6) M and 40 +/- 4% in response to 10(-4) M isoproterenol (n = 11). In the group in which blood was placed on the parietal cortex, pial arterioles did not dilate significantly in response to hypercapnia (8 +/- 3%, n = 11) or hypotension (2 +/- 5%, n = 13) but dilated normally in response to isoproterenol (25 +/- 5% in response to 10(-6) M and 36 +/- 7% in response to 10(-4) M, n = 13). We conclude that prolonged contact of pial arterioles with extravascular blood selectively attenuates cerebrovascular dilation in piglets.     

Adenosine receptors and the nucleoside transporter in human brain vasculature

Evidence suggests that adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels. Adenosine and other nucleosides are also transported across the blood-brain barrier via a saturable, carrier-mediated mechanism. Using direct ligand binding methods, we studied the two adenosine receptor subtypes, A1 and A2 and the nucleoside transporter moiety in human brain microvessels, pial vessels, choroid plexus, and cerebral cortex membranes. The following specific tritiated ligands were used: cyclohexyladenosine (CHA) for A1 receptors; 5'-N-ethylcarboxamide adenosine (NECA) for A2 receptors; nitrobenzylthioinosine (NBMPR) and dipyridamole (DPY) for nucleoside transporters. We find that cerebral microvessels, pial vessels, and choroid plexus have few, if any, A1 receptors, in contradistinction to cerebral membranes, which have a 10-20-fold higher density of A1 receptor sites. Specific high-affinity NECA binding to A2 receptors in cerebral microvessels, pial vessels, and choroid plexus was saturable and was equivalent to that of cerebral cortical membranes. The Bmax and Kd of the high-affinity NECA binding to vessel preparations were approximately 1.3 pmol/mg protein and approximately 250 nM, respectively, which is similar to our previous findings in the rat and pig. NBMPR and DPY binding were also saturable and were consistent with a single class of high-affinity binding sites. The density of nucleoside transporters was approximately four-fold higher in cerebral microvessels than in cerebral cortex, pial vessels, and choroid plexus. These results suggest that human cerebral microvessels have A2, but not A1, receptors and are particularly enriched with the adenosine transporter moiety.     

Adenosine receptor agonists inhibit the release of γ-aminobutyric acid (GABA) from the ischemic rat cerebral cortex

The effects of CPA (a selective A1 receptor agonist), NECA (a mixed A1 and A2 receptor agonist), and CGS 21680 (a selective A2 receptor agonist) on the ischemia-evoked release of gamma-aminobutyric acid (GABA) from rat cerebral cortex was investigated with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. In control animals, superfusate GABA increased from a basal level of 206 +/- 26 nM (mean +/- S.E.M., n = 18) to 10,748 +/- 3,876 nM during the reperfusion period. Pretreatment with adenosine receptor agonists failed to affect basal levels of GABA release. However, CPA (10(-10) M), NECA (10(-9) M), and CGS 21680 (10(-8) M) significantly suppressed the ischemia-evoked release of GABA. The ability to block the ischemia-evoked release of GABA was not evident when the adenosine receptor agonists were administered at higher concentrations. Thus, the selective activation of either A1 or high-affinity A2a adenosine receptors results in an inhibition of ischemia-evoked GABA release.     

Effect of 2-chloroadenosine on cerebrovascular reactivity to hypercapnia in newborn pig.

The effect of local administration of vasodilative concentrations of the adenosine receptor agonist 2-chloroadenosine (2-CADO) on the hyperemic responses of the pial and parenchymal microcirculations to graded hypercapnia was determined. The cranial window and brain microdialysis-hydrogen clearance techniques were utilized in two groups of isoflurane-anesthetized newborn pigs to measure changes in pial diameters and local CBF, respectively, in response to graded hypercapnia in the absence and presence of 2-CADO. Progressive size-dependent dilations of pial arterioles [small = 41 +/- 7 microns (mean +/- SD), intermediate = 78 +/- 13 microns, and large = 176 +/- 57 microns in diameter] occurred in response to graded hypercapnia alone (PaCO2 = 58 and 98 mm Hg) and to superfusions of 2-CADO (10(-5) M) during normocapnia; the magnitude of the dilative response to each of these stimuli was inversely proportional to vessel size. When hypercapnia was induced concomitantly with 2-CADO superfusion, the dilative effects of each stimulus were directly additive. Similarly, local microdialysis infusion of 10(-5) M 2-CADO, which doubled CBF during normocapnia, did not affect the hyperemic response of the parenchymal circulation to graded hypercapnia (PaCO2 = 69 and 101 mm Hg). Our findings are consistent with the participation of adenosine in the mediation of cerebral hypercapnic hyperemia. If, however, adenosine is not involved in this dilative response, our results indicate that concomitant vascular and neuromodulatory actions induced by adenosine receptor stimulation do not affect the mechanism responsible for the hypercapnic hyperemic response.     

Mechanisms of impaired endothelium-dependent cerebral vasodilatation in response to bradykinin in hypertensive rats

Bradykinin produces less dilatation of pial arterioles in stroke-prone spontaneously hypertensive rats than in normotensive Wistar-Kyoto rats. The goals of this study were to determine the mediator of bradykinin-induced dilatation in cerebral arterioles of rats and to determine whether responses to this mediator are altered in hypertensive rats. Diameter of pial arterioles (20-65 microns) was measured using intravital microscopy in 18 normotensive and 17 hypertensive rats. Superfusion of 3 x 10(-7) M bradykinin dilated pial arterioles by 53 +/- 4% (mean +/- SEM) in normotensive rats but only 33 +/- 6% in hypertensive rats (p less than 0.05 versus normotensive rats). Vasodilatation in response to bradykinin was almost completely inhibited by 280 units/ml catalase in both normotensive and hypertensive rats (n = 7 and n = 7, respectively) whereas 150 units/ml superoxide dismutase (n = 6 and n = 5, respectively) and 1 mM deferoxamine (n = 5 and n = 5, respectively) did not attenuate bradykinin-induced vasodilatation. These findings suggest that hydrogen peroxide is the mediator of bradykinin-induced dilatation in cerebral arterioles of rats. We also examined responses of cerebral arterioles to hydrogen peroxide in five normotensive and six hypertensive rats. Dilator responses of cerebral arterioles to 3.2 x 10(-5) M to 1.6 x 10(-4) M hydrogen peroxide did not differ in normotensive and hypertensive rats, which suggests that impaired dilatation of cerebral arterioles in response to bradykinin is not related to altered responsiveness of smooth muscle to an endothelium-derived relaxing factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine stimulates cAMP-mediated taurine release from LRM55 glial cells

The possible role of adenosine as a modulator or transmitter in the central nervous system was tested by measuring its effects on LRM55 astroglial cells. Two related cellular responses were measured--receptor activated increases in intracellular cAMP and cAMP-mediated taurine release. Taurine is a neuroinhibitory amino acid that is taken up, stored, and released from primary cultures of astrocytes and astroglial cells. Three-minute incubations of cells with adenosine caused a dose-dependent accumulation of intracellular cAMP and release of the taurine (EC50 = 5.0 x 10(-5) M and 1.6 x 10(-6) M, respectively). That the cellular responses were mediated through the activation of specific adenosine receptors was indicated by the observations that the adenosine receptor antagonist isobutylmethylxanthine (IBMX) but not the beta-adrenergic receptor antagonist 1-propranolol inhibited responses to adenosine. The study of various adenosine analogs showed a rank order of potency (chloroadenosine = 5'-(N-ethyl)carboxamido-adenosine greater than N6-(L-2-phenylisopropyl)-adenosine greater than cyclohexyladenosine = cyclopentyladenosine) characteristic of the low affinity A2-type adenosine receptors that have been associated with cAMP elevation in several tissues. These results indicate that, in addition to directly affecting neurons, adenosine may have a primary site of action on astroglial cells resulting in taurine release and subsequent inhibition of neuronal activity.     

Contractile responses to endothelin in feline cortical vessels in situ

In this study in chloralose-anaesthetised cats, the vasomotor responses of individual pial vessels on the cortical surface to perivascular subarachnoid microapplication of endothelin were examined. Endothelin (3 x 10(-10) -3 x 10(-6) M) induced marked vasoconstriction of pial arterioles (-33.5 +/- 3.8% at 3 x 10(-6) M) and pial veins (-35.1 +/- 2.7% at 3 x 10(-6) M). The concentration of endothelin inducing half-maximal response was in the nanomolar range, with pial veins being slightly more sensitive to the peptide than pial arterioles. Vasoconstrictions induced by endothelin were extremely prolonged, persisting for approximately 90 min after a single microapplication. Arterioles constricted by endothelin remained responsive to perivascular microapplication of K+ (10 mM) or alkalotic CSF (pH 7.48).     

Responses of rat basilar artery to acetylcholine and platelet products in vivo

Studies in vitro suggest that the basilar artery has distinctive responses to endothelium-dependent stimuli. Our first goal was to examine the effects of acetylcholine on diameter of the basilar artery in vivo. Because aggregating platelets may have important effects on cerebral arteries, our second goal was to examine the effects on the basilar artery of products that are released by platelets (thromboxane, serotonin, and adenosine 5'-diphosphate). Diameter of the basilar artery was measured through a cranial window in anesthetized rats (n = 25). Baseline diameter of the basilar artery was 247 +/- 10 microns mean +/- SEM. Topical application of acetylcholine at 10(-6) and 10(-5) M dilated the basilar artery by 13 +/- 2% and 19 +/- 2%, respectively. The thromboxane analogue U46619 at 10(-8) and 10(-7) M reduced the diameter of the basilar artery by 18 +/- 5% and 29 +/- 4%, respectively. At 10(-8) and 10(-7) M, serotonin had little effect on pial arterioles on the cerebrum but constricted the basilar artery by 18 +/- 2% and 29 +/- 4%, respectively. At 10(-6) and 10(-5) M, adenosine 5'-diphosphate produced marked dilatation of pial arterioles on the cerebrum (9 +/- 2% and 20 +/- 3%, respectively) but had little effect on the basilar artery (increased diameter by 4 +/- 2% and 6 +/- 2%, respectively). Thus, in contrast to some studies of the basilar artery in vitro, acetylcholine produces dilatation of the basilar artery in vivo. Potent constrictor responses to thromboxane and serotonin, in combination with the minimal dilator effect of adenosine 5'-diphosphate, suggest that release of these products during platelet aggregation would favor constriction of the basilar artery.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

The effects of dipyridamole and theophylline on rat pial vessels during hypocarbia

Hypocarbia results in an increase in brain adenosine concentrations, presumably because of brain hypoxia associated with hypocarbic vasoconstriction. It was hypothesized that adenosine limits the degree of hypocarbic vasoconstriction. To test this hypothesis, the effects of dipyridamole and theophylline on CO2 reactivity during hypocarbia were investigated in anesthetized rats. Dipyridamole should reduce the vasoconstriction by potentiating adenosine action, whereas theophylline should increase the vasoconstriction by blocking adenosine receptors. Cortical pial arterioles of mechanically ventilated and anesthetized rats were displayed on a video monitor system through a closed cranial window. Arterial blood pressure and oxygen tension were stable. CO2 reactivity, formulated as 100 X [delta diameter (micron)/resting diameter (micron)]/delta PaCO2 (mmHg), in the hypocarbic phase was calculated before and after topical superfusion of dipyridamole (10(-6) M; n = 7) and theophylline (5 X 10(-5) M; n = 6). CO2 reactivity was significantly decreased after superfusion of dipyridamole (0.57 +/- 0.08; mean +/- SEM) as compared with mock cerebrospinal fluid (CSF) (0.97 +/- 0.17, p less than 0.05, n = 7). On the other hand, CO2 reactivity after superfusion of theophylline was increased (1.63 +/- 0.28) as compared with mock CSF (1.00 +/- 0.20, p less than 0.05, n = 6), indicating that adenosine is involved in hypocarbic vasoconstriction.     

Perivascular blood attenuates noradrenergic but not cholinergic effects on piglet pial arterioles

We examined the chronic and acute effects of perivascular blood on cerebrovascular responses to norepinephrine and acetylcholine in 35 piglets. In the chronic experiment, fresh autologous blood (n = 15) or cerebrospinal fluid (n = 14, control) was placed under the dura mater over the parietal cortex, and the piglets were allowed to recover from anesthesia. One to 4 days later, a closed cranial window was placed over the parietal cortex and baseline pial arteriolar responses and responses to topical application of the neurotransmitters norepinephrine (10(-6) and 10(-4) M) and acetylcholine (10(-4) M) were determined. We also sampled cerebrospinal fluid from under the window during baseline conditions and during application of the neurotransmitters, and we measured the concentrations of prostanoids (6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin F2 alpha, and prostaglandin E2) via radioimmunoassay. Pial arterioles in the chronic control group (n = 13) constricted by 20 +/- 2% (mean +/- SEM) in response to 10(-4) M norepinephrine and by 28 +/- 2% in response to 10(-4) M acetylcholine. In the chronic blood group (n = 14), pial arterioles did not constrict significantly in response to 10(-4) M norepinephrine but constricted normally (23 +/- 4%) in response to 10(-4) M acetylcholine. In the acute experiment, six other piglets had blood placed on the brain surface for 30 minutes and then removed; pial arterioles constricted by 21 +/- 1% in response to 10(-4) M norepinephrine (n = 5) and by 28 +/- 4% in response to 10(-4) M acetylcholine (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous norepinephrine constricts cerebral arterioles via alpha 2-adrenoceptors in newborn pigs

The purpose of this study was to determine whether exogenous norepinephrine mediates cerebrovascular constriction via alpha 1- or alpha 2-adrenoceptors in anesthetized neonatal pigs. Diameters of pial arterioles in anesthetized piglets, 1--6 days old, were investigated using a "closed" cranial window. We examined constrictor effects of norepinephrine on pial arterioles in the absence and presence of relatively selective alpha 1-(prazosin) and alpha 2-(yohimbine) adrenoceptor antagonists (1 mg/kg i.v.). Yohimbine and prazosin inhibited pial arteriolar constriction induced by topical application of clonidine and phenylephrine (10(-6) and 10(-4) M, respectively), and yohimbine did not affect the response to topical phenylephrine. In one group diameter was 188 +/- 13 (mean +/- SEM) micron during control and 146 +/- 12 micron during 10(-5) M norepinephrine (22 +/- 5% constriction). Following yohimbine the same vessels did not constrict significantly. In another group 10(-5) M norepinephrine constricted arterioles by 22 +/- 5%, and this response was unaffected by prazosin (24 +/- 5% constriction). We conclude that pial arterioles are responsive to both alpha 1- and alpha 2-adrenoceptor agonists, that intravenous administration of prazosin and yohimbine results in these drugs crossing the blood-brain barrier and inhibiting constrictor effects of agonists, and that norepinephrine constricts pial arterioles predominantly via alpha 2-adrenoceptors.     

Adenosinergic Modulation of Homocysteine-Induced Seizures in Mice

Homocysteine thiolactone (HTL) elicits seizures in mice at a dose of 850 mg/kg (95-100% of animals) with an average latency time of 19.5 min. These seizures are reversed by both 5' N-ethylcarboximide adenosine (NECA) and flunitrazepam, with respective ED50 doses of 0.025 and 0.20 mg/kg. NECA was approximately four-fold more potent as an inhibitor of HTL-induced seizures than of seizures induced by pentylenetetrazol (PTZ, 75 mg/kg). Flunitrazepam was equipotent in both seizure paradigms. The purine precursor 5-amino-4-imidazole carboxamide riboside, (AICAr), although virtually ineffective against PTZ-induced seizures at doses greater than 1 g/kg, was able to inhibit HTL-induced seizures with an ED50 of approximately 350 mg/kg. The anticonvulsant effect of AICAr was dose and time dependent. The anticonvulsant potency of AICAr was increased by simultaneous administration of the adenosine uptake blocker Mioflazine, whereas the central nervous system (CNS)-impermeable adenosine uptake blocker dipyridamole had no effect. The ability of AICAr to permeate the blood-brain barrier (BBB) is limited (less than 1%) and may explain its low potency as an anticonvulsant. AICAr also has very low potency at brain adenosine A1 and A2 receptors as well as adenosine uptake sites (IC50 greater than 10(-3) M), suggesting that its anticonvulsant properties are not mediated by direct action at these sites. The results indicate that AICAr does have frank anticonvulsant effects and further suggest that HTL-induced seizures may represent a useful paradigm for evaluation of adenosinergic agents. AICAr or more potent derivatives thereof may represent a new class of anticonvulsants with the ability to target seizure foci selectively.     

Histamine elicits competing endothelium-dependent constriction and endothelium-independent dilation in vivo in mouse cerebral arterioles

We used television microscopy and an image-splitting technique to monitor the changes in diameter produced by histamine applied locally to mouse pial arterioles in vivo. A high dose (50 micrograms/ml, 3 X 10(-4) M) of histamine constricted the arterioles, whereas lower doses (20 and 10 micrograms/ml) relaxed them. Constriction was blocked and dilation occurred when selective injury of the endothelium was produced by light from a helium-neon laser in the presence of intravascular Evans blue. From this we conclude that the constriction was endothelium-dependent and was caused by the release of an endothelium-derived constricting factor. Constriction was also blocked by each of two antagonists of the H1 histamine receptor and by pretreatment of the arterioles with indomethacin. H1 blockade unmasked a dilating action of 1 micrograms/ml histamine, a dose too low to affect the diameter of arterioles not treated with the H1 blocker. An H2 blocker interfered with the relaxation by low-dose (10 micrograms/ml, 6 X 10(-5) M) histamine. These data indicate that for mouse pial arterioles, histamine can interact with H1 receptors on the endothelium to release an endothelium-derived constricting factor that causes constriction of the underlying muscle while simultaneously interacting with H2 receptors in the muscle that mediate relaxation of the vessel.