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.     

Specific ouabain binding to brain microvessels and choroid plexus

The energy-dependent transport of ions across the blood-brain barrier and the blood-cerebrospinal fluid barrier by Na+, K+-ATPase is credited with an important role in brain homeostasis. In this study, we have assessed the relative enrichment of Na+, K+-ATPase in regional brain capillary preparations and in the choroid plexus by the quantitative determination of the cardiac glycoside binding sites in these preparations using [3H]ouabain as a ligand. We find that ouabain binds specifically to brain microvessels of the rat and the pig and to the choroid plexus of the pig in a saturable manner. The maximum density of ouabain binding sites in brain microvessels of both species is about one-fourth that of the crude membranes of the cerebrum and cerebellum. The density of ouabain binding sites in the pig choroid plexus is intermediate between that of the brain and brain microvessels. We do not find regional differences in ouabain binding to membrane fractions of the cerebrum and cerebellum, nor any significant differences in ouabain binding to cerebral and cerebellar microvessels. These findings provide quantitative estimates of Na+, K+-ATPase in brain capillaries and choroid plexus.     

Heterogeneity of binding sites for N-ethylcarboxamido[3H]adenosine in rat brain: effects of N-ethylmaleimide

Binding sites for N-ethylcarboxamido[3H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(-)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [3H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A1-adenosine receptor. At concentrations of displacing agent higher than 1 microM, R-PIA was unable to displace [3H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [3H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A2-adenosine receptor. In striatum, however, R-PIA was also unable to displace [3H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A1- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [3H]NECA binding completely. The remaining sites showed IC50 values of 0.13 and 3.68 microM for NECA and R-PIA, respectively. These A2-like [3H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [3H]NECA binding sites, an A1-site, an A2-site and a third, unidentified R-PIA-insensitive site.     

The glucose transporter of the human brain and blood-brain barrier

We identified and characterized the glucose transporter in the human cerebral cortex, cerebral microvessels, and choroid plexus by specific D-glucose-displaceable [3H]cytochalasin B binding. The binding was saturable, with a dissociation constant less than 1 microM. Maximal binding capacity was approximately 7 pmol/mg protein in the cerebral cortex, approximately 42 pmol/mg protein in brain microvessels, and approximately 27 pmol/mg protein in the choroid plexus. Several hexoses displaced specific [3H]cytochalasin B binding to microvessels in a rank-order that correlated well with their known ability to cross the blood-brain barrier; the only exception was 2-deoxy-D-glucose, which had much higher affinity for the glucose transporter than the natural substrate, D-glucose. Irreversible photoaffinity labeling of the glucose transporter of microvessels with [3H]cytochalasin B, followed by solubilization and polyacrylamide gel electrophoresis, labeled a protein band with an average molecular weight of approximately 55,000. Monoclonal and polyclonal antibodies specific to the human erythrocyte glucose transporter immunocytochemically stained brain blood vessels and the few trapped erythrocytes in situ, with minimal staining of the neuropil. In the choroid plexus, blood vessels did not stain, but the epithelium reacted positively. We conclude that human brain microvessels are richly endowed with a glucose transport moiety similar in molecular weight and antigenic characteristics to that of human erythrocytes and brain microvessels of other mammalian species.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

Ontogeny of adenosine uptake sites in guinea pig brain: differential profile of [3H]nitrobenzylthioinosine and [3H]dipyridamole binding sites

The ontogenetic profile of adenosine uptake sites was investigated in guinea pig cerebral cortex and cerebellum using as ligand probes the uptake inhibitors, [3H]nitrobenzylthioinosine ([3H]NBI) and [3H]dipyridamole ([3H]DPR). In cerebral cortex [3H]NBI binding was highest at E50 and decreased subsequently until P28 while in cerebellum after a first peak at E50 and a subsequent decline it increased again until P28. [3H]DPR binding increased by 25% from E40 to P28 in cerebral cortex while in cerebellum hardly any binding could be detected before E50 and it afterwards increased by more than 250% until P28. Scatchard analysis demonstrated that [3H]NBI labeled approximately as many sites as [3H]DPR in cerebral cortex at E44 while at P28 [3H]DPR labeled more than double as many sites. Accordingly, NBI was more potent in displacing [3H]DPR binding at E44 than at P28. These findings suggest that part of the [3H]DPR binding sites, i.e. the NBI-insensitive one develops later than [3H]NBI binding sites during ontogeny in guinea pig cerebral cortex and cerebellum.     

Atrial natriuretic peptide receptors in cerebral microvessels and choroid plexus of spontaneously hypertensive rats

Binding sites of atrial natriuretic peptide in the cerebral microvessels and choroid plexus of spontaneously hypertensive rats (SHR) and Wistar Kyoto control rats (WKY) were measured. In the microvessels, the number of ANP binding sites was lower in SHR than WKY, but there was no difference in affinity of binding sites for the ligand between SHR and WKY. In the choroid plexus, the number of ANP binding sites was lower and the affinity for the ligand was higher in SHR than in WKY. These results suggest that the physiological function regulated by the ANP receptors in the cerebral microvessels and choroid plexus were altered in hypertension and that ANP receptors in the cerebral microvessels and choroid plexus were differentially regulated.     

Heterogeneity of binding sites for N-ethylcar☐amido[H]adenosine in rat brain: Effects of N-ethylmaleimide

Binding sites for N-ethylcar☐amido[³H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(−)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [³H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A₁-adenosine receptor. At concentrations of displacing agent higher than 1 μM, R-PIA was unable to displace [³H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [³H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A₂-adenosine receptor. In striatum, however, R-PIA was also unable to displace [³H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A₁- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [³H]NECA binding completely. The remaining sites showed IC₅₀ values of 0.13 and 3.68 μM for NECA and R-PIA, respectively. These A₂-like [³H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [³H]NECA binding sites, an A₁-site, an A₂-site and a third, unidentified R-PIA-insensitive site.     

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) inhibition of [3H]N-ethylcarboxamidoadenosine (NECA) binding allows the visualization of putative non-A1 adenosine receptors

The binding of the adenosine receptor agonists, [3H]N-ethylcarboxamidoadenosine (NECA) and [3H]cyclohexyladenosine (CHA) to membrane preparations and to cryostat sections of the rat brain was examined. The xanthine derivative, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was ca. 500-fold more effective at A1 than at A2 sites. [3H]CHA binding to A1 adenosine receptors was virtually eliminated by the inclusion of DPCPX (50 nM), while [3H]NECA binding was only partially inhibited. The pattern of DPCPX-insensitive [3H]NECA binding sites was strikingly different from that of A1 receptors and is believed to represent an association with A2 type adenosine receptors and perhaps another or several, previously undescribed non-A1 sites.     

Effects of ischemia on regional ligand binding to adrenoceptors in the rat brain.

Changes in ligand binding to adrenoceptors ([3H]prazosin to alpha 1-receptors, [3H]idazoxan to alpha 2-receptors and [125I]cyanopindolol to beta-receptors) following transient cerebral ischemia were investigated using autoradiographic methods. The binding was quantified in brain sections from control rats, rats subjected to 15 min of 2-vessel occlusion ischemia, and rats with recirculation times of 1 h, 1 week or 4 weeks after ischemia. No significant change in alpha 1-receptor binding was observed during and immediately following ischemia, but a decrease was noted in the vulnerable hippocampal CA1 region following 1 week's survival. In the parietal cortex, the ligand binding to alpha 1-receptors increased at 4 weeks. A reduced [3H]idazoxan binding was observed 1 h after ischemia in the temporal cortex and amygdala. No change in ligand binding to beta-receptors was seen in the early phase postischemia, but a marked increase had occurred in the hippocampal CA1 region at 1 and 4 weeks after ischemia (+163% and +142%, respectively), presumably due to accumulation of macrophages expressing beta-receptors. The early postischemic changes in receptor binding may represent downregulation of the adrenoceptors by processes activated during ischemia, while neuronal degeneration, compensatory mechanisms in surviving neurons and proliferation of non-neuronal cells may account for the subsequent changes.     

Serotonin-1C sites in the choroid plexus are not linked in a stimulatory or inhibitory way to adenylate cyclase

The association of the serotonin recognition sites in the pig choroid plexus (5-HT-1C sites) with an adenylate cyclase was examined. The interaction of serotonin and mianserin with [3H]mesulergine binding was not affected by the stable GTP analogue GppNHp. The binding of [3H]serotonin to choroid plexus membranes was also unaffected by GppNHp while a dose-dependent decrease was observed in pig cortical and hippocampal membranes. The porcine choroid plexus contained a forskolin- and histamine-sensitive adenylate cyclase. Serotonin, however, was ineffective in this preparation. While forskolin-stimulated adenylate cyclase in the rat hippocampus was inhibited by serotonin forskolin-stimulated adenylate cyclase in the choroid plexus was insensitive to serotonin. These results indicate that the serotonin recognition sites in the choroid plexus are not linked in a stimulatory or inhibitory way to an adenylate cyclase, in contrast with other 5-HT-1 receptor subtypes.     

Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-HT1C and 5-HT2 recognition sites

The presence, pharmacological properties and anatomical distribution of serotonin-1C and serotonin-2 receptor subtypes were studied in the human brain by both radioligand binding and autoradiographic procedures. Frontal cortex, hippocampus and choroid plexus from human brains obtained at autopsy without history of neurological diseases were used in this study. [3H]5-HT and [3H]mesulergine were used to label 5-HT1C recognition sites while [3H]ketanserin was used to label 5-HT2 receptors. The pharmacological profile of 5-HT1C sites which are very concentrated in the choroid plexus, was extremely similar to that of pig and rat 5-HT1C sites. These receptors were also detected in the hippocampus and the cortex from human brain. The general distribution of 5-HT1C sites in human and rat brain was similar although slight differences were observed. Human 5-HT2 receptors were concentrated in cortical areas but also found in the hippocampus. The pharmacological profile of these receptors was extremely similar in human and pig brain tissue, but differed in certain respects to that found in rat brain 5-HT2 receptors. The anatomical distribution of 5-HT2 receptors is similar in human and rat brain with some differences at the microscopic level. The importance of species differences in the development of 5-HT2 compounds is discussed.     

Retrograde axonal transport of β-adrenoreceptors in rat brain: Effect of reserpine

Retrograde axonal transport of β-adrenoreceptors was assessed by measuring the accumulation of binding sites for the β-receptor ligand [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICP) distal to a unilateral 6-hydroxydopamine (6-OHDA) lesion placed in the ascending noradrenergic axons of the locus coeruleus. Accumulation of binding sites was linear over a 3 day period and was blocked by intracerebroventricular 6-OHDA given 1 day prior to sacrifice. A single dose of reserpine (5 mg/kg, i.p.) caused a long lasting (6–8 week) biphasic depletion of frontal cortex norepinephrine (NE) associated with increased frontal cortex binding of another β-receptor ligand. [³H]dihydroalprenolol ([³H]DHA), at 7–14 days, and again at 28 days post-reserpine. Unlike the changes in cortical β-receptors, retrograde transport of [¹²⁵I]ICP in presynaptic noradrenergic neurons was decreased or blocked completely at 7–14 days and at 6 weeks, and was increased to 470% and 240% of control at 21 days and 8 weeks after reserpine. Anterograde transport of [³H]DHA binding sites was measured by accumulation proximal to a 6-OHDA lesion in this pathway. This transport varied in a pattern similar to that seen for retrograde transport of [¹²⁵I]ICP binding sites. These data and others suggest that presynaptic β-receptors are regulated independently of frontal cortex β-receptors, which appear to be located primarily on postsynaptic cells. On the other hand, the regulation of both anterograde and retrograde transport appears to be interrelated since both types of transport were altered in a similar way in the face of long-term NE depletion by reserpine.     

Autoradiographic demonstration of peripheral adenosine binding sites using [3H]NECA

Autoradiographic localization of 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding sites revealed a heterogeneous labeling of guinea-pig intestine with heavy labeling over the enteric ganglia and in clusters over the mucosa; a low level of label was homogeneously distributed over the muscularis externa. Under the conditions employed, no binding sites were revealed using [3H]-N6-cyclohexyladenosine ([3H]CHA), although both [3H]CHA and [3H]NECA binding sites were localized over comparable areas of rat brain. The relationship of the [3H]NECA binding sites to extracellular adenosine receptors is discussed.     

Adenosine uptake and [3H]nitrobenzylthioinosine binding in developing rat brain

The ontogenesis of adenosine transport sites as labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [3H]NBI binding in newborn and adult rats were reflected by changes in Bmax and not Kd values. As a measure of the utility of [3H]NBI as a probe for identifying functional adenosine transport sites, we examined [3H]NBI binding to and [3H]adenosine accumulation by intact brain cells prepared from adult and newborn rats. For [3H]NBI binding to brain cells from adult rats, the values of Kd were 0.092 nM and of Bmax were 274 fmol/mg protein. For newborns, slightly higher Kd and Bmax values were observed; 0.2 nM and 395 fmol/mg protein, respectively. [3H]Adenosine accumulation was higher in brain cells from one-day-old than from adult rat brains. Kinetically this uptake was best described by a two-component model: the Vmax values for the high- and low-affinity uptake, and the Km value for the high-affinity component in one-day-old rats were greater than in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and regional distribution of serotonin S2-receptors in human brain

[³H]Ketanserin binding was characterized in vitro in human brain homogenates and the regional distribution of the sites was determined.In human brain, [³H]ketanserin was found to bind on serotonin (5-HT) S₂-receptors; only 5-HT antagonists competed with the labelled ligand at nanomolar concentrations; other drugs were much less active or inactive. Special attention was paid to the choice of a displacer, here methysergide, to determine the blank value (non-displaceable binding). [³H]Ketanserin binding in human brain displayed similar binding characteristics to the S₂-receptor in the rat frontal cortex, high affinity (K_d 0.69 nM) and relatively slow dissociation rate.The regional distribution of serotonin S₂-receptors labelled with [³H]ketanserin was studied in 30 different regions of human brain. The highest number of receptors was measured in the cortex. However, within the cortex the distribution was also inhomogeneous, a much lower number of sites being found in the pre- and post-central gyri. In the dopaminergic areas and the cerebellum the number of sites was quite low, and only few binding sites were detected in the corpus callosum, the medulla and the hypophysis.The large number of serotonin S₂-receptors in the human cortex suggests that serotonin has an important role in this brain region.     

3H]SCH39166, a D1 dopamine receptor antagonist: binding characteristics and localization.

Schering-Plough Research has developed a new, more specific analogue of SCH23390. This compound, SCH39166, has been shown to be a potent, specific, D1 receptor antagonist with several features which are advantageous over its predecessor. In this report, the binding characteristics of [3H]SCH39166 are described by in vitro analysis in rat brain tissues. The binding was shown to be of high affinity (Kd in the low nM range), saturable, and specific (readily displaceable with SCH23390, but not with the D2 receptor antagonists sulpiride or haloperidol). The binding of SCH39166 is more selective for binding to D1 receptors than SCH23390 with regard to overlap of the latter compound onto 5HT2 and 5HT1C receptors. Autoradiographic localization of D1 receptor sites labeled with [3H]SCH39166 showed a very specific distribution in areas known to contain high quantities of D1 receptors. These regions included the deepest layer of the cerebral cortex, the caudate-putamen, nucleus accumbens, olfactory tubercle, entopeduncular nucleus, and substantia nigra-pars reticulata, as well as less dense binding in a few other areas. At the concentration of ligand used (1 nM), there was a noticeable paucity of labeling in lamina IV of the cerebral cortex and in the choroid plexus, regions of high 5HT2 and 5HT1C receptor binding, respectively. Thus, SCH39166 represents a new D1 receptor antagonist which shows a greater specificity for the D1 receptor than its predecessor SCH23390. As previously shown, another distinct advantage of this compound is its stability in primates which should allow the determination of the effects and utility of D1 receptor antagonism in vivo.     

Ontogenesis of adenosine receptors in the central nervous system of the rat

The ontogeny of adenosine receptors was studied in rat brain and spinal cord using the specific ligand [3H]cyclohexyladenosine [( 3H]CHA). The [3H]CHA affinity constant (Kd) and the maximum receptor binding capacity (Bmax) were analyzed at all ages and in all CNS regions studied. Throughout development the Kd of [3H]CHA binding remained relatively stable and for cortex, cerebellum, subcortex, midbrain, brainstem and spinal cord ranged from 2.2 +/- 0.2 to 5.5 +/- 0.6 nM (mean +/- S.E.M.). In contrast, the Bmax values from 1- and 90-day animals increased by as little as 2-fold in subcortical regions and by as much as 9- and 16-fold in cortex and cerebellum, respectively. The highest density of binding sites was observed in subcortical structures and the lowest in brainstem and midbrain. In cortex, a steady increase in receptor number began at day 1 and stopped at the adult level by 21 days. In cerebellum, maximum receptor proliferation began at about 14 days and continued to adulthood. Other CNS regions showed intermediate rates of receptor development. These differences may reflect both the pattern of postnatal neurogenesis in the rat CNS and the maturation of those neural elements containing adenosine receptors.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Bradykinin receptors of cerebral microvessels stimulate phosphoinositide turnover.

We examined by ligand binding methods whether bradykinin (BK) receptors exist in rat and pig cerebral microvessels, and in the cerebral cortex from which the microvessels were isolated. We found a high-affinity and saturable BK receptor site in both rat and pig cerebral microvessels, but not in their cerebral cortex. The maximal density of binding and the dissociation constant were 8.0 +/- 4.1 and 6.8 +/- 1.5 fmol/mg of protein and 47 +/- 24 and 150 +/- 8 pM (mean +/- SD) in cerebral microvessels of the pig and rat, respectively. The high-affinity specific binding of BK was effectively displaced by des-Arg0[Hyp3-Thi5-8,D-Phe7]BK, a specific B2 receptor antagonist, but not by des-Arg9[Leu8]BK, a specific B1 antagonist. We also demonstrated that BK increases phosphatidylinositol hydrolysis in cerebral microvessels of the rat and pig. This effect was also blocked by the B2, but not by the B1, antagonist. Increased phosphatidylinositol hydrolysis was manifested by a rapid transient increase in inositol trisphosphate and the later slow accumulation of inositol bisphosphate and inositol monophosphate. Preincubation of microvessels with phorbol ester, stable GTP analogs, pertussis toxin, or in Ca(2+)-free buffer did not influence BK activation of phosphatidylinositol hydrolysis. These results demonstrate the existence of BK receptors of the B2 subtype in brain microvessels, which may play an important role in modulation of the brain microcirculation, probably via increased phosphoinositide turnover.