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Object Cerebrovascular dysfunction after subarachnoid hemorrhage (SAH) may contribute to ischemia, but little is known about the contribution of intracerebral arterioles. In this study, the authors tested the hypothesis that SAH inhibits the vascular reactivity of intracerebral arterioles and documented the time course of this dysfunction. Methods Subarachnoid hemorrhage was induced using an endovascular filament model in halothane-anesthetized male Sprague-Dawley rats. Penetrating intracerebral arterioles were harvested 2, 4, 7, or 14 days postinsult, cannulated using a micropipette system that allowed luminal perfusion and control of luminal pressure, and evaluated for reactivity to vasodilator agents. Results Spontaneous tone developed in all pressurized (60 mm Hg) intracerebral arterioles harvested in this study (from 66 rats), with similar results in the sham and SAH groups. Subarachnoid hemorrhage did not affect dilation responses to acidic pH (6.8) but led to a persistent impairment of endothelium-dependent dilation responses to adenosine triphosphate (p < 0.01), as well as a transient attenuation (p < 0.05) of vascular smooth muscle–dependent dilation responses to adenosine, sodium nitroprusside, and 8-Br-cyclic guanosine monophosphate (cGMP). Impairment of NO-mediated dilation was more sustained than adenosine- and 8-Br-cGMP–induced responses (up to 7 days postinsult compared with 2 days). All smooth muscle–dependent responses returned to sham levels by 14 days after SAH. Conclusions Subarachnoid hemorrhage led to a persistent impairment of endothelium-dependent dilation and a transient attenuation of vascular smooth muscle–dependent dilation responses to adenosine. Impairment of NOmediated dilation occurred when the response to cGMP was intact, suggesting a change in cGMP levels rather than an alteration in intracellular mechanisms downstream from cGMP.

Adenosine (ADO) is a potent cerebral vasodilator and has been proposed as a metabolic regulator of cerebral blood flow. However, the signal transduction pathway by which ADO causes vasodilation in cerebral microvessels is currently unknown. The current study was designed to investigate the role of cyclic nucleotides and cyclic nucleotide-dependent protein kinases in ADO-induced dilation of resistance-sized rat cerebral arterioles that develop spontaneous tone. Arterioles were cannulated and perfused intraluminally at constant flow (2 microl/min) and pressure (60 mm Hg). ADO (29.7 +/- 2.0%; 1 microM), CGS-21680 (16 +/- 4%, 1 microM), 8-bromo-cyclic guanosine monophosphate (8 Br-cGMP; 29.9 +/- 3.9%; 100 microM), sodium nitroprusside (SNP; 30.6 +/- 3.3%, 1 microM), cyclic guanine monophosphate-dependent protein kinase activator (Sp-8-pCPT-cGMPS, 25.9 +/- 4.2%; 10 microM), forskolin (30.5 +/- 5.9%; 0.1 microM), and pH 6.8 all produced large dilations. The selective cGMP-dependent protein kinase inhibitor, Rp-8-pCPT-cGMPS (10 microM), had no effect on resting diameter or reactivity to acidic pH, but significantly ( < 0.05) attenuated arteriolar dilations to ADO (59%, n = 8), CGS-21680 (60%, n = 4), SNP (62%, n = 3), 8 Br-cGMP (88%, n = 3), and Sp-8-pCPT-cGMPS (98%, n = 3). H8, the less-selective cyclic nucleotide-dependent protein kinase inhibitor, had similar effects as Rp-8-pCPT-cGMPS. Additionally, the inhibitor of the soluble guanylate cyclase, 1H-[1,24]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), blocked the response to SNP (70% inhibition) and significantly inhibited the ADO response (43% inhibition). In contrast, inhibition of the cyclic ADO monophosphate (cAMP)-dependent protein kinase Rp-8-CPT-cAMPS had no effect on the ADO, SNP, or pH responses, but significantly blocked forskolin-induced vasodilation (53%). It is concluded that ADO-induced vasodilation in cerebral microvessels, at least in part, involves cGMP and cGMP-dependent protein kinase, but not cAMP or cAMP-dependent kinase. Our data therefore provides a new insight into mechanisms by which ADO invokes vasodilation in cerebral microvascular arterioles.

1. The mammalian brain ventricles are lined with ciliated ependymal cells. As yet little is known about the mechanisms by which neurotransmitters regulate cilia beat frequency (CBF). 2. Application of 5-HT to ependymal cells in cultured rat brainstem slices caused CBF to increase. 5-HT had an EC50 of 30 microM and at 100 microM attained a near-maximal CBF increase of 52.7 +/- 4.1 % (mean +/- s.d.) (n = 8). 3. Bathing slices in Ca2+-free solution markedly reduced the 5-HT-mediated increase in CBF. Fluorescence measurements revealed that 5-HT caused a marked transient elevation in cytosolic Ca2+ ([Ca2+]c) that then slowly decreased to a plateau level. Analysis showed that the [Ca2+]c transient was due to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores; the plateau was probably due to extracellular Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels. 4. Application of ATP caused a sustained decrease in CBF. ATP had an EC50 of about 50 microM and 100 microM ATP resulted in a maximal 57.5 +/- 6.5 % (n = 12) decrease in CBF. The ATP-induced decrease in CBF was unaffected by lowering extracellular [Ca2+], and no changes in [Ca2+]c were observed. Exposure of ependymal cells to forskolin caused a decrease in CBF. Ciliated ependymal cells loaded with caged cAMP exhibited a 54.3 +/- 7.5 % (n = 9) decrease in CBF following uncaging. These results suggest that ATP reduces CBF by a Ca2+-independent cAMP-mediated pathway. 5. Application of 5-HT and adenosine-5'-O-3-thiotriphosphate (ATP-gamma-S) to acutely isolated ciliated ependymal cells resulted in CBF responses similar to those of ependymal cells in cultured slices suggesting that these neurotransmitters act directly on these cells. 6. The opposite response of ciliated ependymal cells to 5-HT and ATP provides a novel mechanism for their active involvement in central nervous system signalling.

Although fluctuations in cytosolic Ca2+ concentration have a crucial role in relaying intracellular messages in the cell, the dynamics of Ca2+ storage in and release from intracellular sequestering compartments remains poorly understood. The rapid release of stored Ca2+ requires large concentration gradients that had been thought to result from low-affinity buffering of Ca2+ by the polyanionic matrices within Ca2+-sequestering organelles. However, our results here show that resting luminal free Ca2+ concentration inside the endoplasmic reticulum and in the mucin granules remains at low levels (20-35 microM). But after stimulation, the free luminal [Ca2+] increases, undergoing large oscillations, leading to corresponding oscillations of Ca2+ release to the cytosol. These remarkable dynamics of luminal [Ca2+] result from a fast and highly cooperative Ca2+/K+ ion-exchange process rather than from Ca2+ transport into the lumen. This common paradigm for Ca2+ storage and release, found in two different Ca2+-sequestering organelles, requires the functional interaction of three molecular components: a polyanionic matrix that functions as a Ca2+/K+ ion exchanger, and two Ca2+-sensitive channels, one to import K+ into the Ca2+-sequestering compartments, the other to release Ca2+ to the cytosol.

Endothelial cells mediate the actions of a variety of vasoactive substances, including ATP. ATP vasodilatatory actions have been shown to depend on a calcium-dependent release of endothelium-derived relaxing factor(s) (EDRF). ATP induced a vasodilatation of pial penetrating microvessels when applied intraluminally; these relaxations were mediated by the endothelium and followed release of nitric oxide (NO), since they were sensitive to blockade of NO-synthesizing enzymes by NG-nitro-L-arginine (1 mM) and NG-mono-methyl-L-arginine (0.1 mM). We have also investigated the electrophysiological actions of extracellular ATP on rat brain microvascular (RBMEC) and bovine aortic endothelial cells (BAEC) using the patch-clamp technique. While BAEC were hyperpolarized by ATP (10 microM), ATP caused the activation of a depolarizing nonselective cation current in brain endothelial cells. NO production measurements by [3H]citrulline assay and by direct amperometric determination also revealed that after exposure to 1-100 microM ATP, RBMEC released NO. NO release from RBMEC was abolished by removal of external calcium. We conclude that, in the brain, ATP exerts its vasoactive roles by altering the electrophysiological properties of endothelial cells by acting on receptor-operated ion channels, thus providing a mechanism for calcium entry and subsequent release of EDRF.

Background and Purpose— Disturbances in cerebral arteriolar function, in addition to large vessel vasospasm, may be responsible for ischemia after subarachnoid hemorrhage. The purpose of this study was to test the hypothesis that subarachnoid hemorrhage alters cerebral microvascular reactivity. Methods— An endovascular filament model was used to induce subarachnoid hemorrhage in halothane-anesthetized male Sprague-Dawley rats. We evaluated pial arteriolar responses to sciatic nerve stimulation, topically applied vasoactive agents (adenosine or sodium nitroprusside), and CO 2 inhalation in rats subjected to subarachnoid hemorrhage at 1 to 5 days after insult. Results— In sham-operated rats, sciatic nerve stimulation evoked a 23.5±1.8% increase in arteriolar diameter, which was significantly attenuated to 13.7±0.9%, 12.8±2.5%, and 18.8±2.9% at 24, 48, and 72 hours after subarachnoid hemorrhage, respectively ( P P 2 reactivity was unaffected by subarachnoid hemorrhage. Conclusions— Pial arteriolar responses to cortical activation may be decreased in the initial 2 to 3 days after experimental subarachnoid hemorrhage.

Background and Purpose— Conducted vasomotor responses likely play an important role in cerebrovascular regulation, but it is unclear how these responses may be affected by ischemia. The purpose of this study was to evaluate the hypothesis that cerebral ischemia and reperfusion (I/R) alters vascular conduction in cerebral arterioles. Methods— Middle cerebral artery occlusion (MCAO) was induced by an intraluminal filament technique in 4 groups of rats: (A) 2-hour MCAO/24-hour reperfusion (n=14); (B) 2-hour MCAO/1-hour reperfusion (n=7); (C) 1-hour MCAO/24-hour reperfusion (n=6); and (D) 1-hour MCAO/1-hour reperfusion (n=5). Neurological status and infarction (2,3,5-triphenyltetrazolium chloride staining) were evaluated after I/R. Conducted vasomotor responses were assessed in intracerebral branches of the MCA, by following the longitudinal spread of vasodilation or vasoconstriction to localized microapplication of ATP or adenosine. Results— Local microapplication of ATP evoked a biphasic constriction (17±3%) and dilation (7±2%) response, whereas adenosine elicited only dilation (11±2%). These local responses spread longitudinally along sham-control arterioles (1 mm conduction distance) with rapid spatial decay. Ischemia followed by 24-hour reperfusion (groups A and C) led to a marked potentiation of conducted dilation responses: dilation to ATP conducted with virtually no decay in I/R arterioles. Augmentation of conductivity was not observed in the 1-hour reperfusion groups (B and D). Moreover, I/R did not alter conducted constriction. Conclusions— Ischemia-reperfusion led to a specific augmentation of conducted vasodilation in cerebral arterioles. Presumably, enhanced conductivity may improve cerebral perfusion after ischemia.

Despite caffeine's wide consumption and well-documented psychoactive effects, little is known regarding the effects of caffeine on neurovascular coupling. In the present study, we evaluated the effects of caffeine, an adenosine receptor antagonist, on intracerebral arterioles in vitro and subsequently, on the pial circulation in vivo during cortical activation induced by contralateral sciatic nerve stimulation (SNS). In our in vitro studies, we utilized isolated intracerebral arterioles to determine the effects of caffeine (10 or 50 μmol/L) on adenosine-induced vasodilatation. At the lower concentration, caffeine was without effect, but at the higher concentration, caffeine produced significant attenuation. In our in vivo studies, we determined the cerebrospinal fluid (CSF) caffeine concentrations at 15, 30, and 60 mins after intravenous administration of 5, 10 and 40 mg/kg. At the latter two concentrations, CSF levels exceeded 10 μmol/L. We then evaluated the pial arteriolar response during cortical activation caused by contralateral SNS after administering caffeine intravenously (0, 5, 10, 20 30, and 40 mg/kg). The pial circulation was observed through a closed cranial window in chloralose-anesthetized Sprague—Dawley rats. The contralateral sciatic nerve was isolated, positioned on silver electrodes and stimulated for 20 secs (0.20 V, 0.5 ms, and 5 Hz). Arteriolar diameter was quantified using an automated video dimension analyzer. Contralateral SNS resulted in a 23.8%±3.9% increase in pial arteriolar diameter in the hindlimb sensory cortex under control conditions. Intravenous administration of caffeine at the lowest dose studied (5 mg/kg) had no effect on either resting arteriolar diameter or SNS-induced vasodilatation. However, at higher doses (10, 20, 30, and 40 mg/kg, intravenously), caffeine significantly ( P