Cerebral Microvascular Smooth Muscle Research Articles

Epoxyeicosatrienoic Acid Inhibits the Apoptosis of Cerebral Microvascular Smooth Muscle Cells by Oxygen Glucose Deprivation via Targeting the JNK/c-Jun and mTOR Signaling Pathways.

As a component of the neurovascular unit, cerebral smooth muscle cells (CSMCs) are an important mediator in the development of cerebral vascular diseases such as stroke. Epoxyeicosatrienoic acids (EETs) are the products of arachidonic acid catalyzed by cytochrome P450 epoxygenase. EETs are shown to exert neuroprotective effects. In this article, the role of EET in the growth and apoptosis of CSMCs and the underlying mechanisms under oxygen glucose deprivation (OGD) conditions were addressed. The viability of CMSCs was decreased significantly in the OGD group, while different subtypes of EETs, especially 14,15-EET, could increase the viability of CSMCs under OGD conditions. RAPA (serine/threonine kinase Mammalian Target of Rapamycin), a specific mTOR inhibitor, could elevate the level of oxygen free radicals in CSMCs as well as the anti-apoptotic effects of 14,15-EET under OGD conditions. However, SP600125, a specific JNK (c-Jun N-terminal protein kinase) pathway inhibitor, could attenuate oxygen free radicals levels in CSMCs as well as the anti-apoptotic effects of 14,15-EET under OGD conditions. These results strongly suggest that EETs exert protective functions during the growth and apoptosis of CSMCs, via the JNK/c-Jun and mTOR signaling pathways in vitro. We are the first to disclose the beneficial roles and underlying mechanism of 14,15-EET in CSMC under OGD conditions.

The protective effect of epoxyeicosatrienoic acids on cerebral ischemia/reperfusion injury is associated with PI3K/Akt pathway and ATP-sensitive potassium channels.

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolite of arachidonic acid, have been demonstrated to have neuroprotective effect. Phosphatidylinositol 3-kinase (PI3K)/Akt and ATP-sensitive potassium (KATP) channels are thought to be important factors that mediate neuroprotection. However, little is known about the role of PI3K/Akt and KATP channels in brain after EETs administration. In vitro experiment, oxygen-glucose deprivation (OGD) was performed in cultured rat cerebral microvascular smooth muscle cells (SMCs) for 4 h. The effect of 14,15-EET on OGD induced cell apoptosis was examined after reoxygenation. Western blot and real-time PCR were used to analyze the expression of Kir6.1, SUR2B (two subunits of KATP channels) and p-Akt on cerebral microvascular SMCs. In vivo experiments, we use 12-(3-adamantan-1-yl-ureido)-dodecanoic acid [AUDA, a specific soluble epoxide hydrolase (sEH) inhibitor] to confirm the effect of EETs indirectly. Rats were injected intraperitoneally with AUDA before being subjected to middle cerebral artery occlusion (MCAO). We detected the apoptosis and the expression of p-Akt, Kir6.1 and SUR2B in ischemic penumbra. The results showed that EETs protect against cerebral ischemia/reperfusion (I/R) injury and upregulated the expression of p-Akt and Kir6.1 in both of ischemic penumbra and OGD induced cerebral microvascular SMCs. The protective effect was inhibited by Wortmannin (a specific PI3K inhibitor) and Glib (a specific KATP inhibitor) respectively in vitro experiment. In conclusion, these results suggested that the protective effect of EETs on cerebral I/R injury is associated with PI3K/Akt pathway and KATP channels. Furthermore, the PI3K pathway may contribute to mediating KATP channels on cerebral microvascular SMCs.

Effects of Anoxia and Hypoxia on Amyloid Precursor Protein Processing in Cerebral Microvascular Smooth Muscle Cells

Cerebral amyloid angiopathy (CAA) is characterized by the degeneration of cerebral microvascular smooth muscle cells (MV-SMC) and the replacement of normal vessel wall components by beta-amyloid (Abeta) protein. Little is known regarding the mechanisms of SMC degeneration in CAA. The effects of anoxia on the metabolism of the amyloid precursor protein (APP) were studied to investigate the MV-SMC response to anoxic stress and its possible role in the pathogenesis of CAA. MV-SMC exposed to chronic anoxia (24-48 hours) showed a decrease in expression of the 2 putative alpha-secretase enzymes, mature TACE (TNFalpha-converting enzyme) and ADAM10 (a disintegrin and metalloprotease). A concomitant decrease in the alpha-secretase cleavage products sAPPalpha and C83 was observed. Investigation of mRNA expression showed an increase in TACE and a sharp decrease in ADAM10 at 24 hours. Exposing MV-SMC to hypoxia (1% O2) revealed a different pattern of expression with no significant change in TACE protein, but an increase in TACE mRNA occurring at a later time point (48 hours). There was no change in ADAM10 mRNA expression, but a reduction in mature ADAM10 with a parallel increase in immature ADAM10 protein. These results demonstrate a requirement for oxygen in the regulation of the alpha-secretase pathway during APP metabolism.

ω-Oxidation of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Cerebral Microvascular Smooth Muscle and Endothelium by Alcohol Dehydrogenase 4

20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.

The Oxygen Radical Scavenger Pyrrolidine Dithiocarbamate Enhances Interleukin-1β-Induced Cyclooxygenase-2 Expression in Cerebral Microvascular Smooth Muscle Cells

Oxidative stress and inducible cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) formation have been proposed to play an important role in cytokine-induced vascular pathology. To explore the relationship between oxidative stress and COX-2 induction, cultured murine cerebral microvascular smooth muscle cells (SMCs) were stimulated with interleukin-1β (IL-1β) in the presence or absence of an oxygen radical scavenger, pyrrolidine dithiocarbamate (PDTC). IL-1β increased COX-2 protein expression in a dose- and time-dependent manner, an increase that was further enhanced by PDTC in a dose-dependent manner. PDTC did not, however, affect the expression of COX-1 protein. In the presence of 100 μM PDTC, PGE 2 production induced by IL-1β (5 ng/ml) was increased by threefold as compared with IL-1β alone. Although PDTC enhanced COX-2 protein expression, it did not increase IL-1β-induced expression of COX-2 mRNA, indicating that the regulatory effect occurred at the posttranscriptional level. The time course of COX-2 protein degradation indicated that PDTC also did not alter the stability of the COX-2 protein induced by IL-1β. These results suggest that endogenous oxygen radicals may blunt COX-2 induced by IL-1β through an effect on translation.

Mechanism of permissive prostacyclin action in cerebrovascular smooth muscle

Prostacyclin permissively allows increased cAMP and cerebral vasodilation to hypercapnia in piglets. The prostacyclin receptor (IP) is coupled to phospholipase C (PLC) in piglet cerebral microvascular smooth muscle cells (SMC). We hypothesize that inhibition of PLC blocks the permissive action of IP receptor agonist, iloprost, and direct activation of PKC substitutes for the IP receptor agonist in SMC. SMC cAMP production was measured at normal pHi/pHo and with reduced pHi/pHo in the absence and presence of iloprost (100 pM). Half of the cells were pretreated with U73122, the PLC inhibitor, which decreased the basal IP 3 and blocked the increase in IP 3 caused by iloprost. Without iloprost, decreasing pHi/pHo increased cAMP production (40%). With iloprost, the cAMP response to acidosis increased to over 80%. U73122 prevented accentuation of the cAMP response by iloprost. Phorbol myristate acetate augmented the response to acidosis similarly to iloprost. These data suggest IP agonists augment the cAMP response to acidosis via coupling through PLC to activate PKC.

Phospholipase C activation by prostacyclin receptor agonist in cerebral microvascular smooth muscle cells.

The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.

CAMP production by piglet cerebral vascular smooth muscle cells: pH(o), pH(i), and permissive action of PGI(2).

In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI(2). Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI(2), hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI(2)-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pH(i) (11 +/- 6 vs. 32 +/- 10% in the absence and presence of 10(-10) M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 +/- 9 vs. 41 +/- 5% in the presence of 10(-12) and 10(-9) M iloprost, respectively). Rapid decreases in pH(i) stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pH(i). We conclude that PGI(2) receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI(2) does not involve inhibition of a pertussis toxin-sensitive G protein.

PHo, pHi, and PCO2 in stimulation of IP3 and [Ca2+]c in piglet cerebrovascular smooth muscle.

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pHo) cause increases in Ins(1,4,5)P3 and cytosolic calcium ([Ca2+]c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pHo 7. 4, PCO2 36 mm Hg), metabolic alkalosis (pHo 7.7, PCO2 36 mm Hg), or respiratory alkalosis (pHo 7.7, PCO2 19 mm Hg). Intracellular pH (pHi) and [Ca2+]c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P3 was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pHi from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P3, as we showed previously. Metabolic and respiratory alkalosis increased [Ca2+]c about 80% and 110%, respectively. Neither Ins(1,4,5)P3 nor [Ca2+]c increased in cells treated with aCSF that produced control pHo with increased pHi (7.3). In contrast, when pHo increased (7.7), but pHi was maintained at control (7.2), Ins(1,4,5)P3 increased from 123 pmol/well to 307 pmol/well and [Ca2+]c increased 46%. However, the increase of [Ca2+]c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P3 with resultant elevation in [Ca2+]c. While the Ins(1,4,5)P3 signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.

Posttranslational regulation of cyclooxygenase by tyrosine phosphorylation in cerebral endothelial cells.

Endothelium-derived cyclooxygenase (COX) products regulate cerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebral microvessels. We investigated the role of protein phosphorylation in the regulation of COX activity. The protein tyrosine phosphatase (PTP) inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acid rapidly stimulated COX activity, whereas the protein tyrosine kinase inhibitors, genistein and tyrphostins, inhibited it. Protein synthesis inhibitors did not reverse the stimulation of COX activity evoked by PTP inhibitors. Similar changes were observed in other vascular cells from newborn pigs that also express COX-1 and COX-2 (cerebral microvascular smooth muscle cells and aortic endothelial cells) but not in human umbilical vein endothelial cells or Swiss 3T3 fibroblasts that express COX-1 only. Tyrosine-phosphorylated proteins were immunodetected in endothelial cell lysates. COX-2 immunoprecipitated from 32P-loaded endothelial cells incorporated 32P that was increased by PTP inhibitors. COX-2, but not COX-1, was detected in endothelial fractions immunoprecipitated with anti-phosphotyrosine. These data indicate that tyrosine phosphorylation posttranslationally regulates COX activity in newborn pig vascular cells and that COX-2 is a substrate for phosphorylation.

Prostaglandin production in cultured cerebral microvascular smooth muscle is serum dependent.

To expand the understanding of cerebrovascular eicosanoid metabolism, the ability of smooth muscle isolated from murine cerebral microvessels to produce prostaglandins (PGs) was studied in vitro. Cultures from SJL and BALB/c mice produced primarily prostaglandin E2 (PGE2) and I2 (PGI2) in response to exogenous arachidonate and calcium ionophore as well as the agonists acetylcholine and epinephrine. Subconfluent smooth muscle cultures demonstrated a two- to threefold increased capacity to produce PG compared with confluent cultures. In contrast, serum deprivation of smooth muscle caused an 80-90% diminution in both PGE2 and PGI2 production but had no effect on PG release in cerebromicrovascular endothelium. Reintroduction of serum to smooth muscle restored PG production within 6h, and the restoration was inhibited by 1 microM dexamethasone. Message for both prostaglandin H synthase (PGHS)-1 and -2 was detectable in smooth muscle grown in the presence of serum, but PGHS-2 message was not present in serum-deprived cultures. Furthermore, readdition of serum induced a massive increase in PGHS-2 mRNA with only a small increase in PGHS-1 message. The serum induction of PGHS-2 was corroborated by immunohistochemistry and Western blotting. Thus cerebromicrovascular smooth muscle may contribute significantly to the formation of PG under circumstances likely to be present during central nervous system pathologies. The induction of PGHS, particularly PGHS-2, may play a key role in this process.

Molecular Characterization of an Arachidonic Acid Epoxygenase in Rat Brain Astrocytes

Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P450 (P450) epoxygenase to epoxyeicosatrienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ current in vascular smooth muscle cells from large cerebral arteries. Because of the close association between astrocytes and the cerebral microcirculation, we hypothesized that brain epoxygenase activity originates from astrocytes. This study was designed to identify and localize an AA epoxygenase in rat brain astrocytes. We also tested the effect of EETs on whole-cell K+ current in rat cerebral microvascular smooth muscle cells. A functional assay was used to demonstrate endogenous epoxygenase activity of intact astrocytes in culture. Oligonucleotide primers derived from the sequence of a known hepatic epoxygenase, P450 2C11, were used in reverse transcription/polymerase chain reaction of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned into a plasmid vector and sequenced. A polyclonal peptide antibody was raised against P450 2C11 and used in Western blotting and immunocytochemical staining of cultured astrocytes. A voltage-clamp technique was used to test the effect of EETs on whole-cell K+ current recorded from rat cerebral microvascular muscle cells. Based on elution time of known standards and inhibition by miconazole, an inhibitor of P450 AA epoxygenase, cultured astrocytes produce 11,12- and 14,15-EETs when incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunoreactivity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staining of cultured astrocytes. EETs enhanced outward K+ current in muscle cells from rat brain microvessels. Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.

Effects of hypercapnia on prostanoid and cAMP production by cerebral microvascular cell cultures.

In the newborn pig cerebral circulation, arteriolar dilation in response to hypercapnia requires the presence of intact endothelium and is accompanied by an indomethacin-sensitive increase in cortical adenosine 3',5'-cyclic monophosphate (cAMP). The effects of short-term hypercapnia on production of dilator prostanoids and cAMP were investigated using newborn pig cerebral microvascular smooth muscle cells and endothelial cells cultured both separately and in noncontact coculture. Microvascular smooth muscle cells respond to hypercapnia (pH 7.00 +/- 0.05 PCO2 75 +/- 3 mmHg) by a 1.3- to 1.7-fold increase in basal cAMP production that is not affected by indomethacin, whereas hypercapnia and 80 mM sodium propionate do not affect iloprost-stimulated cAMP production. Microvascular endothelial cells cultured on Millicel inserts respond to hypercapnia by a two- to fourfold increase in prostacyclin (as 6-keto-prostaglandin F1 alpha) and prostaglandin E2 production in both luminal and abluminal compartments. For noncontact coculture, Millicel inserts with endothelial cells (as hypercapnia-sensitive producers of prostanoids) were installed into cell culture dishes with aspirin-pretreated smooth muscle cells (as targets for endothelium-derived dilator prostanoids). Exposure of noncontact microvascular cell cocultures to hypercapnia results in a three- to fourfold stimulation of prostanoid and cAMP production. Therefore, short-term hypercapnia increases cAMP production by microvascular smooth muscle cells via 1) a direct (prostanoid independent) mechanism and 2) an endothelial-dependent pathway that involves prostanoids. Endothelium-produced prostanoid signals are necessary for a full increase in cAMP production by cerebral microvascular smooth muscle cells in response to hypercapnia.

VASCULAR SMOOTH MUSCLE SIGNALS UPREGULATE ENDOTHELIAL CYCLOOXYGENASE EXPRESSION IN THE NEWBORN PIG CEREBRAL CIRCULATION. † 1403

Cellular communication between vascular endothelium and smooth muscle are important in the regulation of vascular function. In the newborn cerebral circulation, endothelium-derived cyclooxygenase products participate in vascular smooth muscle responsiveness to many physiological stimuli. Our present data indicate that vascular smooth muscle, in turn, may influence functions of vascular endothelium. Endothelial cells from the newborn pig cerebral microvessels cultured separately or in coculture with microvascular smooth muscle cells were used to investigate a possible role of cellular communication in the regulation of endothelial cyclooxygenase expression. Microvascular endothelial cells (freshly isolated as well as confluent and quiescent primary cultures) constitutively express both cyclooxygenase isoforms, COX-1 and COX-2, as detected by Western blots and immunostaining with the isozyme-specific antibodies. Results with the isozyme-selective inhibitor NS-398 suggest that COX-2 provides a large portion of endothelial prostanoid production. Endothelial cells grown in non-contact coculture with smooth muscle cells for 24-48 hr show induction of COX-1 and COX-2 proteins. There are a 10-50-fold increases in endogenous prostanoid accumulation and cyclooxygenase activity that are inhibited by dexamethasone, cycloheximide and actinomycin D. Similar changes are induced in endothelial cells exposed to smooth muscle cell-conditioned media. These results indicate that cerebral microvascular smooth muscle cells produce a diffusable compound(s) that upregulate endothelial cyclooxygenase thus modifying endothelial cell function.

Low CO2 stimulates inositol phosphate turnover and increased inositol 1,4,5-trisphosphate levels in piglet cerebral microvascular smooth muscle cells.

In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-Ins[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 +/- 0.7 and 4.1 +/- 0.8 cpm/micrograms protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 +/- 1.8 and 38 +/- 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 +/- 16 and 64.1 +/- 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.

Arginine Vasopressin Stimulates Protein Synthesis but Not Proliferation of Cultured Vascular Endothelial Cells

We previously showed that rat and cow blood vessels contain arginine vasopressin (AVP) of local origin. In the present study, to investigate potential roles for this locally produced peptide, we examined the effects of AVP on growth of cultured vascular endothelial and smooth muscle cells (EC, SMC). Treatment of cultured bovine aortic endothelial (BAE), murine cerebral microvascular endothelial (MME), or murine cerebral microvascular smooth muscle (MMSM) cells with AVP produced dose-dependent increases in total protein content with maximum increases of 11, 46, and 33% over vehicle-treated controls, respectively. AVP also dose-dependently increased new protein synthesis, as reflected by [3H]leucine incorporation into BAE and MME cell protein, with maximal increases of 34 and 24%, respectively. In addition, the AVP-stimulated increase in protein synthesis could be significantly attenuated in both BAE and MME cells by simultaneous addition of a specific AVP V1 receptor antagonist. In contrast, AVP did not affect total cell number or DNA synthesis, as reflected by [3H]thymidine incorporation, in any cell type. Neither was cell size, as measured by forward light scatter with a fluorescence-activated cell sorter (FACS), changed by treatment with AVP. However, the density of individual cells, as measured by orthogonal light scatter with FACS, was increased by AVP. These data demonstrate that AVP stimulates protein synthesis but not proliferation of cultured vascular EC and SMC and suggest that locally produced AVP may function as a growth factor for these cells.

Prostanoid synthesis in response to high CO2 in newborn pig brain microvascular endothelial cells

Hypercapnia-induced cerebral vasodilation involves prostanoids in newborn pigs. However, the source of prostanoids has not been determined. The current study was designed to address the hypothesis that piglet cerebral microvascular endothelial cells increase their synthesis of prostanoids in response to high CO2. Microvascular endothelial cells, smooth muscle cells, and glia were isolated and grown in primary culture. They were identified morphologically and by indirect immunofluorescence staining. Cerebral microvascular endothelial cell cultures from newborn pigs produced equal amounts of 6-ketoprostaglandin (PG) F1 alpha (stable hydrolysis product of PGI2), PGE2 and a small amount of PGF2 alpha under basal conditions. Administration of calcium ionophore A23187 to the endothelial cells increased release of all three prostanoids in a dose- and time-dependent manner. Exposure of piglet cerebral microvascular endothelial cells to higher than normal CO2 increased the production of 6-keto-PGF1 alpha and PGE2 but not of PGF2 alpha. The enhanced prostanoid biosynthesis was concentration dependent, peaking at 14% CO2, and was detected during the first 10 min exposure to 14% CO2. Hypercapnia-induced increased synthesis of prostanoids was blocked dose dependently by the simultaneous addition of PGH synthase inhibitor indomethacin. High CO2 did not increase prostanoid production by cerebral microvascular smooth muscle cells or glia, although A23187 enhanced prostanoid formation by both cell types. These data show that high CO2 stimulates prostanoid synthesis by newborn pig cerebral microvascular endothelial cells, which is consistent with an involvement of cerebral vascular endothelium in hypercapnia-induced vasodilation.

Rat cerebral microvascular smooth muscle cells in culture.

This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.

Cerebral microvascular smooth muscle in tissue culture.

Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco's modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a "hill and valley" pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-35]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, alpha-actin. The identity of alpha-actin is confirmed by analysis of NH2-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of alpha-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation.