Characterization of culture from smooth muscle cells isolated from rat middle cerebral arteries.

Although human brain represents only 2% of the body mass, it uses around 20 % of the organism energy. Due to the brain's limited energy storage, the oxygen and glucose necessary to support brain functions depends on the correct blood supply. The main components of the arteries are smooth muscle cells, which are considered the main regulators of vascular tone and blood flow distribution. The information currently available on the functioning of the cerebral arteries and their cell constituents is extremely scarce. Thus, the aim of this work was to develop an in vitro model of smooth muscle cells derived from rat middle cerebral artery. Explants were collected from rat middle cerebral artery and adhered to collagen-coated culture dishes. Immunocytochemical analysis showed that the cells present in the culture expressed α-actin, a protein characteristic of the contractile phenotype of these cells. In addition, these cells did not express the endothelial marker, vWF. To evaluate the functionality of these cells the response to contractile agents, serotonin and noradrenaline, and to relaxing agent, sodium nitroprusside was determine by Planar Cell Surface Area analysis. Together the data obtained show that the cell culture obtained through the procedure described resulted in cells presenting the markers characteristic of smooth muscle cells and maintaining the usual contractile response, indicating that the cells obtained through this may be used as a model for characterization and study of functional behavior of the middle cerebral artery, as well as interaction studies between vascular and neuronal system.

Sex differences in the structure and function of rat middle cerebral arteries.

Epidemiological studies demonstrate that there are sex differences in the incidence, prevalence, and outcomes of cerebrovascular disease (CVD). The present study compared the structure and composition of the middle cerebral artery (MCA), neurovascular coupling, and cerebrovascular function and cognition in young Sprague-Dawley (SD) rats. Wall thickness and the inner diameter of the MCA were smaller in females than males. Female MCA exhibited less vascular smooth muscle cells (VSMCs), diminished contractile capability, and more collagen in the media, and a thicker internal elastic lamina with fewer fenestrae compared with males. Female MCA had elevated myogenic tone, lower distensibility, and higher wall stress. The stress/strain curves shifted to the left in female vessels compared with males. The MCA of females failed to constrict compared with a decrease of 15.5 ± 1.9% in males when perfusion pressure was increased from 40 to 180 mmHg. Cerebral blood flow (CBF) rose by 57.4 ± 4.4 and 30.1 ± 3.1% in females and males, respectively, when perfusion pressure increased from 100 to 180 mmHg. The removal of endothelia did not alter the myogenic response in both sexes. Functional hyperemia responses to whisker-barrel stimulation and cognition examined with an eight-arm water maze were similar in both sexes. These results demonstrate that there are intrinsic structural differences in the MCA between sexes, which are associated with diminished myogenic response and CBF autoregulation in females. The structural differences do not alter neurovascular coupling and cognition at a young age; however, they might play a role in the development of CVD after menopause.NEW & NOTEWORTHY Using perfusion fixation of the middle cerebral artery (MCA) in calcium-free solution at physiological pressure and systematically randomly sampling the sections prepared from the same M2 segments of MCA, we found that there are structural differences that are associated with altered cerebral blood flow (CBF) autoregulation but not neurovascular coupling and cognition in young, healthy Sprague-Dawley (SD) rats. Understanding the intrinsic differences in cerebrovascular structure and function in males and females is essential to develop new pharmaceutical treatments for cerebrovascular disease (CVD).

Distribution and Migration of Human Placental Mesenchymal Stromal Cells in the Brain of Healthy Rats after Stereotaxic or Intra-Arterial Transplantation.

Human placenta mesenchymal stromal cells were injected to healthy rats either stereotaxically into the striatum or intra-arterially through the internal carotid artery. Some cells injected into the brain migrated along the corpus callosum both medially and laterally or concentrated around small blood vessels. A small fraction of MSC injected intra-arterially adhered to the endothelium and stayed inside blood vessels for up to 48 hours mostly in the basin of the middle cerebral artery. Neither stereotaxic, nor intra-arterial transplantation of mesenchymal stromal cells modulated the proliferation of neural stem cells in the subventricular zone of the brain, but stereotaxic transplantation suppressed activation of their proliferation in response to traumatization with the needle.

Large conductance voltage- and calcium-gated potassium channels (BK) in cerebral artery myocytes of perinatal fetal primates share several major characteristics with the adult phenotype.

Large conductance voltage- and calcium-gated channels (BK) control fundamental processes, including smooth muscle contractility and artery diameter. We used a baboon (Papio spp) model of pregnancy that is similar to that of humans to characterize BK channels in the middle cerebral artery and its branches in near-term (165 dGa) primate fetuses and corresponding pregnant mothers. In cell-attached patches (K+pipette = 135 mM) on freshly isolated fetal cerebral artery myocytes, BK currents were identified by large conductance, and voltage- and paxilline-sensitive effects. Their calcium sensitivity was confirmed by a lower Vhalf (transmembrane voltage needed to reach half-maximal current) in inside-out patches at 30 versus 3 µM [Ca2+]free. Immunostaining against the BK channel-forming alpha subunit revealed qualitatively similar levels of BK alpha protein-corresponding fluorescence in fetal and maternal myocytes. Fetal and maternal BK currents recorded at 3 µM [Ca2+]free from excised membrane patches had similar unitary current amplitude, and Vhalf. However, subtle differences between fetal and maternal BK channel phenotypes were detected in macroscopic current activation kinetics. To assess BK function at the organ level, fetal and maternal artery branches were pressurized in vitro at 30 mmHg and probed with the selective BK channel blocker paxilline (1 µM). The degree of paxilline-induced constriction was similar in fetal and maternal arteries, yet the constriction of maternal arteries was achieved sooner. In conclusion, we present a first identification and characterization of fetal cerebral artery BK channels in myocytes from primates. Although differences in BK channels between fetal and maternal arteries exist, the similarities reported herein advance the idea that vascular myocyte BK channels are functional near-term, and thus may serve as pharmacological targets during the perinatal-neonatal period.

Intracellular acidification alters myogenic responsiveness and vasomotion of mouse middle cerebral arteries.

Intracellular pH (pHi) in the vascular wall modulates agonist-induced vasocontractile and vasorelaxant responses in mesenteric arteries, whereas effects on myogenic tone have been unsettled. We studied the role of Na(+),HCO3(-) cotransporter NBCn1 in mouse isolated middle cerebral arteries and the influence of pHi disturbances on myogenic tone. Na(+),HCO3(-) cotransport was abolished in arteries from NBCn1 knockout mice and steady-state pHi ∼0.3 units reduced compared with wild-type mice. Myogenic tone development was low under control conditions but increased on treatment with the NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). This effect of L-NAME was smaller in arteries from NBCn1 knockout than wild-type mice. Myogenic tone with L-NAME present was significantly lower in arteries from NBCn1 knockout than wild-type mice and was abolished by rho-kinase inhibitor Y-27632. The arteries displayed vasomotion, and this rhythmic contractile pattern was also attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or intracellular [Ca(2+)] were seen between arteries from NBCn1 knockout and wild-type mice. We propose that NO production and rho-kinase-dependent Ca(2+) sensitivity are reduced at low pHi in pressurized mouse middle cerebral arteries. This likely impedes the ability to adjust to changes in perfusion pressure and regulate cerebral blood flow.

Theory and applications of geometric scaling of localized calcium release events.

Geometric measures of localized calcium release (LCR) events have been used to understand their biophysical basis. We found power law scaling between three such metrics-maximum amplitude (MA), mass above half-maximum amplitude (MHM), and area at half-maximum amplitude (AHM). In an effort to understand this scaling a minimal analytic model was employed to simulate LCR events recorded by confocal line scan. The distribution of logMHM as a function of logAHM, pMHM(pAHM), was dependent on model parameters such as channel open time, current size, line scan offset, and apparent diffusion coefficient. The distribution of log[MHM/AHM] as a function of logMA, p[MHM/AHM](pMA), was invariant, reflecting the gross geometry of the LCR event. The findings of the model were applied to real LCR line scan data from rabbit portal vein myocytes, rat cerebral artery myocytes, and guinea pig fundus knurled cells. pMHM(pAHM) could be used to distinguish two populations of LCR events in portal vein, even at the scale of "calcium noise," and to calculate the relative current of the two. The relative current was 2. pMHM(pAHM) could also be used to study pharmacological effects. The pMHM(pAHM) distribution of knurled cell LCR events was markedly contracted by ryanodine, suggesting a reduction in channel open time. The p[MHM/AHM](pMA) distributions were invariant across all cell types and were consistent with the model, underlying the common physical basis of their geometry. The geometric scaling of LCR events demonstrated here may help with their mechanistic characterization.

Interstitial cells from rat middle cerebral artery belong to smooth muscle cell type.

It is now established that non-contractile cells with thin filopodia, also called vascular interstitial cells (VICs), are constitutively present in the media of many, if not all, blood vessels. The aim of this study was to determine the type of cell lineage to which arterial VICs belong using immunocytochemical, and real-time and reverse transcription PCR (RT-PCR). Using RT-PCR, we compared gene expression profiles of single VICs and smooth muscle cells (SMCs) freshly dispersed from rat middle cerebral artery. Both VICs and SMCs expressed the SMC marker, smooth muscle myosin heavy chain (SM-MHC), but did not express fibroblast, pericyte, neuronal, mast cell, endothelial or stem cell markers. Freshly isolated VICs also did not express c-kit, which is the marker for interstitial cells of Cajal in the gastrointestinal tract. Immunocytochemical labelling of contractile proteins showed that VICs and SMCs expressed SM-MHC similarly to the same degree, but VICs in contrast to SMCs had decreased expression of alpha-SM-actin and very low or no expression of calponin. Real-time RT-PCR was consistent with immunocytochemical experiments and showed that VICs had four times lower gene expression of calponin comparing to SMCs, which may explain VICs' inability to contract. VICs had greater expression than SMCs of structural proteins such as non-muscular beta-actin and desmin. The results obtained suggest that VICs represent a subtype of SMCs and may originate from the same precursor as SMCs, but later develop filopodia and a non-contractile cell phenotype.

Molecular studies of BKCa channels in intracranial arteries: presence and localization.

Large conductance calcium-activated potassium channels (BK(ca)) are crucial for the regulation of cerebral vascular basal tone and might be involved in cerebral vasodilation relevant to migraine and stroke. We studied the differential gene expression of mRNA transcript levels and protein expression of the BK(Ca) channel in rat basilar, middle cerebral, and middle meningeal arteries by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, and Western blotting. Distribution patterns were investigated using in situ hybridization and immunofluorescence studies. RT-PCR and quantitative real-time PCR detected the expression of the BK(Ca) channel mRNA transcript in rat basilar, middle cerebral, and middle meningeal arteries, with the transcript being expressed more abundantly in rat basilar arteries than in middle cerebral and middle meningeal arteries. Western blotting detected the BK(Ca) channel protein in rat basilar and middle cerebral arteries. In situ hybridization and immunofluorescence studies confirmed that the BK(Ca) channel mRNA and protein expression was localized to smooth muscle cells in all three intracranial arteries. The data thus suggest the presence and localization of both mRNA and protein expression of the BK(Ca) channel in the smooth muscle cell layer in rat basilar, middle cerebral, and middle meningeal arteries.

N-methyl-D-aspartate (NMDA) antagonists--S(+)-ketamine, dextrorphan, and dextromethorphan--act as calcium antagonists on bovine cerebral arteries.

Ketamine, an intravenous anesthetic and a major drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Ketamine's enantiomer, S(+)-ketamine, acts stereoselectively on neuronal NMDA receptors. The purpose of this in vitro study was to compare the direct effects of S(+)-ketamine, 2 other noncompetitive NMDA receptor antagonists (dextrorphan and dextromethorphan), and the calcium entry blocker nimodipine on the cerebral vasculature, using bovine middle cerebral arteries as an experimental model. Arterial rings were mounted in isolated tissue chambers equipped with isometric tension transducers to obtain pharmacologic dose-response curves. In the absence of exogenous vasoconstrictors, the NMDA antagonists or nimodipine had negligible effects on cerebral arterial tone. When rings were preconstricted with either potassium or the stable thromboxane A2 mimetic U46619, the NMDA antagonists and nimodipine each produced dose-dependent relaxation. Prior endothelial stripping had no effect on subsequent drug-induced relaxation of K+-constricted rings. In Ca2+-deficient media containing either potassium or U46619, the NMDA antagonists and nimodipine each produced competitive inhibition of subsequent Cainduced constriction. In additional experiments, arterial strips were mounted in isolated tissue chambers to directly measure calcium uptake, using 45calcium (45Ca) as a radioactive tracer. The NMDA antagonists and nimodipine each blocked potassium-stimulated or U46619-stimulated Ca2+ uptake into arterial strips. These results indicate that S(+)-ketamine, dextrorphan, and dextromethorphan, like nimodipine, directly dilate cerebral arteries by acting as calcium antagonists; they all inhibit 45Ca uptake through both potential-operated (potassium) and receptor-operated (U46619) channels in cerebrovascular smooth muscle.

Embryonic neural stem cells transplanted in middle cerebral artery occlusion model of rats demonstrated potent therapeutic effects, compared to adult neural stem cells.

Cell therapy using stem cells is awaited by stroke patients with impaired movement and cognitive functions, although intravenous alteplase-administration ameliorated outcomes of patients receiving the therapy within 3 h of onset. In this study, we explored the therapeutic effects of neural progenitor cells (NPC) upon middle cerebral artery occlusion (MCAO) model of rats with exploration of the differences between adult and embryonic NPCs in therapeutic effects. GFP-labeled adult or embryonic NPCs were transplanted for transient MCAO model of rats at 1h after reperfusion. Rats were examined behaviorally using limb placement test, rotarod test and cylinder test with neuroradiological assessment using magnetic resonance imaging (MRI). Consequently after euthanasia, rats were immunohistochemically investigated to explore graft survival and immune reaction. MRI of rats receiving NPCs revealed significant reduction of infarct volumes, compared to vehicle-treated rats with corresponding behavioral amelioration. The transplanted cells were surviving in rats receiving NPCs, although the number of embryonic NPCs was significantly higher than that of adult NPCs. Iba-1-positive inflammatory cells of rats receiving adult NPCs were prominent, compared to those receiving embryonic NPCs, which might be a rationale for the differences between rats receiving adult and embryonic NPCs in the number of surviving NPCs. On the contraries, adult NPCs surely demonstrated therapeutic effects with a few surviving cells, thus indicating that the therapeutic effects might be due to trophic/growth factor-secretion from transplanted NPCs, rather than replacement of damaged host neurons. Therapeutic effects of NPCs for MCAO model of rats were clarified in this study. Transplantation of NPCs will be a hopeful strategy for stroke patients, although further studies are required for the patient safety and underlying mechanisms.

Direct regulation of BK channels by phosphatidylinositol 4,5-bisphosphate as a novel signaling pathway.

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)) only through phospholipase C (PLC)-generated PIP(2) metabolites that target Ca(2+) stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP(2) activates BK channels independently of PIP(2) metabolites. PIP(2) enhances Ca(2+)-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP(2) acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP(2) (diC16). The PIP(2)-channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6-S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP(2)-induced activation is drastically potentiated by accessory beta(1) (but not beta(4)) channel subunits. Moreover, PIP(2) robustly activates BK channels in vascular myocytes, where beta(1) subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP(2) effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+beta(1) and PI4-kinaseIIalpha robustly activates BK channels, suggesting a role for endogenous PIP(2) in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP(2) antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP(2) levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP(2) directly activates vascular myocyte BK channels to control vascular tone.

On the presence of neurotrophin p75 receptor on rat sympathetic cerebrovascular nerves.

Although the presence of neurotrophin p75 receptor on sympathetic nerves is a well-recognised feature, there is still a scarcity of details of the distribution of the receptor on cerebrovascular nerves. This study examined the distribution of p75 receptor on perivascular sympathetic nerves of the middle cerebral artery and the basilar artery of healthy young rats using immunohistochemical methods at the laser confocal microscope and transmission electron microscope levels. Immunofluorescence methods of detection of tyrosine hydroxylase (TH) in sympathetic nerves, p75 receptor associated with the nerves, and also S-100 protein in Schwann cells were applied in conjunction with confocal microscopy, while the pre-embedding single and double immunolabelling methods (ExtrAvidin and immuno-gold-silver) were applied for the electron microscopic examination. Immunofluorescence studies revealed "punctuate" distribution of the p75 receptor on sympathetic nerves including accompanying Schwann cells. Image analysis of the nerves showed that the level of co-localization of p75 receptor and TH was low. Immunolabelling applied at the electron microscope level also showed scarce co-localization of TH (which was intra-axonal) and p75. Immunoreactivity for p75 receptor was present on the cell membrane of perivascular axons and to a greater extent on the processes of accompanying Schwann cells. Some Schwann cell processes were adjacent to each other displaying strong immunoreactivity for p75 receptor; immunoreactivity was located on the extracellular sites of the adjacent cell membranes suggesting that the receptor was involved in cross talk between these. It is likely that variability of locations of p75 receptor detected in the study reflects diverse interactions of p75 receptor with axons and Schwann cells. It might also imply a diverse role for the receptor and/or the plasticity of sympathetic cerebrovascular nerves to neurotrophin signalling.

Inhibition of angiogenesis by Abeta peptides.

Abeta peptides are naturally occurring peptides forming beta-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimer's disease. Since several peptides adopting a beta-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Abeta on angiogenesis. We show that in vitro, Abeta dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Abeta peptides containing a higher content of beta-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Abeta dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Abeta dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Abeta on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Abeta potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Abeta delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Abeta is an angiogenesis inhibitor.

Thrombospondin-4 and its variants: expression and differential effects on endothelial cells.

BACKGROUND: In a recent large-scale genetic association study, a single nucleotide polymorphism in the thrombospondin-4 (TSP-4) gene, resulting in a proline-for-alanine substitution at position 387, was associated with a significantly increased risk for premature atherosclerosis. TSP-4 had not previously been implicated in vascular pathology, and very little information is available on its expression and functions. METHODS AND RESULTS: The goal of this study was to assess TSP-4 expression in vessel wall and to identify differences in functions of TSP-4 variants that could account for the proatherogenic effects of the (P387)TSP-4 variant. TSP-4 expression was demonstrated in human endothelial cells (ECs) and vascular smooth muscle cells from brain blood vessels and coronary arteries. (P387)TSP-4 and its fragment (residues 326 to 722), but not the A(387) forms, suppressed EC adhesion and proliferation. The (P387)TSP-4 was more active in inducing the phosphorylation of focal adhesion kinase, consistent with inhibition of proliferation. Both variant fragments increased the proliferation of human aortic smooth muscle cells. CONCLUSIONS: TSP-4 is expressed by vascular cells and influences the vessel wall by modulating the proliferation of ECs and smooth muscle cells. The A387P substitution is a "gain-of-function" mutation, favoring a form of TSP-4 that interferes with EC adhesion and proliferation and may thereby be proatherogenic.

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells.

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.

Effects of prostaglandin F2alpha on membrane currents in rabbit middle cerebral arterial smooth muscle cells.

Although PGF(2alpha) affects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF(2alpha) on membrane potentials, Ca(2+)-activated K(+) (K(Ca)) channels, delayed rectifier K(+) (K(V)) channels, and L-type Ca(2+) channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF(2alpha) significantly hyperpolarized membrane potentials and increased the amplitudes of total K(+) currents. PGF(2alpha) increased open-state probability but had little effect on the open and closed kinetics of K(Ca) channels. PGF(2alpha) increased the amplitudes of K(V) currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF(2alpha) decreased the amplitudes of L-type Ca(2+) currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF(2alpha) on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.

Essential role of Gap junctions in NO- and prostanoid-independent relaxations evoked by acetylcholine in rabbit intracerebral arteries.

BACKGROUND AND PURPOSE: Direct intercellular communication via gap junctions may play a central role in endothelium-dependent relaxations that are mediated by a conducted hyperpolarization and do not involve the synthesis of NO and prostanoids. In the present study, inhibitory peptides homologous to the Gap27 domain of the second extracellular loop of connexin37/connexin43 and connexin40, designated as 37,43Gap27 and 40Gap27, respectively, were used to evaluate the role of this mechanism in intracerebral arteries. METHODS: Isolated rings of rabbit middle cerebral artery were constricted by histamine (10 micromol/L) in the presence of N(G)-nitro-L-arginine methyl ester (300 micromol/L) and indomethacin (10 micromol/L). Concentration-relaxation curves for acetylcholine were constructed in the presence and absence of 37,43Gap27 and 40Gap27. Specific antibodies were used to delineate the distribution of connexin37, connexin40, connexin43, and connexin45 within the arterial wall. RESULTS: Individually, 37,43Gap27 and 40Gap27 minimally affected endothelium-dependent relaxations to acetylcholine at concentrations of 300 micro mol/L, whereas their combination (at 300 micromol/L each) inhibited the maximal response by approximately 70% and increased the EC50 value for relaxation by approximately 15-fold. In endothelium-denuded rings, this peptide combination did not attenuate responses to sodium nitroprusside, an exogenous source of NO. Gap junction plaques, whose incidence was highest in endothelium, were constructed from connexin40 and connexin43 in the media and connexin37, connexin40, and connexin43 in the endothelium. CONCLUSIONS: The findings confirm that direct communication via gap junctions contributes to agonist-induced relaxations of intracerebral arteries. More than one connexin subtype appears to participate in such responses.

Contractile responses and myosin phosphorylation in reconstituted fibers of smooth muscle cells from the rat cerebral artery.

String-shaped reconstituted smooth muscle fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured smooth muscle cells derived from the rat cerebral artery. The fibers contracted in response to KCl, 5-hydroxytryptamine (5-HT), noradrenaline, endothelin-1, endothelin-2, angiotensin II, prostaglandin F2alpha and prostaglandin E2. 5-HT-induced contraction was partially inhibited by the L-type voltage-dependent Ca2+ channel inhibitor nifedipine, putative non-selective cationic channel inhibitor SKF96365 and intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), and completely abolished by the myosin light chain kinase inhibitor ML-9. The fibers pre-contracted by 5-HT were completely relaxed by the Rho kinase inhibitor Y-27632, serine/threonine kinase inhibitor staurosporine, 8-bromo cyclic GMP and papaverine, and partially relaxed by dibutyryl cyclic AMP. Moreover, 5-HT as well as endothelin-1 and KCl enhanced 20-kDa myosin light chain phosphorylation in the fibers. These results suggested that the characteristics of contraction of the fibers reflect typical contractilities of vascular smooth muscle tissues. This technique will allow us to directly address questions relating to heterogeneity of receptor mechanisms and intracellular pathways of vascular smooth muscle contraction as a function of vessel type.

L-Citrulline recycle for synthesis of NO in cerebral perivascular nerves and endothelial cells.

Recycle of L-citrulline to form L-arginine in cerebral perivascular nerves has been well described, providing direct evidence that nitric oxide (NO) is synthesized and released from these nerves to act as the transmitter for vasodilation. NO is also synthesized and released from cerebral endothelial cells, involving L-citrulline conversion to L-arginine. Evidence for the presence of enzymes involved in the conversion, however, has not been shown. The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL), and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in endothelial cells of middle cerebral arteries and the circle of Willis of the pig, therefore, were examined using combined immunohistochemical and histochemical techniques. NOS-, ASS-, and ASL-immunoreactivities were found in almost all endothelial cells of all cerebral arteries examined. All ASS-, ASL-, and NOS-immunoreactive (I) endothelial cells also stained positively for NADPHd, suggesting that ASS, ASL, and NOS were colocalized in endothelial cells of middle cerebral arteries and the circle of Willis. These results provide morphological evidence that cerebral vascular endothelial cells like cerebral perivascular nerves contain enzymes necessary for recycling L-citrulline to L-arginine to synthesize NO via an argininosuccinate (AS) pathway.

Role of endothelium in shear stress-induced constrictions in rat middle cerebral artery.

BACKGROUND AND PURPOSE: Luminal shear stress has been reported to constrict cerebral arteries and arterioles of several species. Although the endothelium is not required for this response, it is not known whether the endothelium enhances or attenuates shear stress-induced constrictions. METHODS: Middle cerebral arteries (MCAs) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized to 80 mm Hg in the absence of luminal flow. In some MCAs, the endothelium was selectively loaded with fura 2 for the measurement of endothelial Ca(2+) concentration. Luminal shear stress was increased by adjusting luminal flow while maintaining a constant intraluminal pressure. RESULTS: After the development of spontaneous tone in MCAs without luminal flow, inside diameters were approximately 190 microm. MCAs constricted approximately 15% when luminal flow was increased to produce a shear stress of 50 dyne/cm(2). The shear stress-induced constrictions were more pronounced in vessels without intact endothelium. Scavenging reactive oxygen species with 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) or superoxide dismutase/catalase significantly inhibited the shear stress-induced constrictions in vessels with intact endothelium and in vessels in which the endothelium had been removed. In intact vessels, endothelial Ca(2+) increased 33 nmol/L (from 133+/-11 to 166+/-12 nmol/L) when shear stress was increased to 50 dyne/cm(2). The presence of N(G)-nitro-L-arginine methyl ester (L-NAME), L-NAME+indomethacin, or L-NAME+indomethacin+charybdotoxin had no significant effect on the shear stress-induced constrictions in MCAs with intact endothelium. CONCLUSIONS: We conclude that the endothelium plays a role in attenuating the shear stress-induced constrictions in rat MCAS: The attenuation does not appear to be by release of NO, prostacyclin, or endothelium-derived hyperpolarizing factor. The endothelium apparently attenuates the constriction by an unknown dilating factor, by a dilating process, or simply by attenuating the mechanical force of the shear stress as it is transmitted to the abluminal side of the vessel.