Regulation of prostaglandin H synthase-2 expression in cerebromicrovascular smooth muscle by serum and epidermal growth factor.

Growth factors may play a role in the formation of prostaglandins (PG) by cerebral blood vessels during development or reaction to injury. In smooth muscle cultures isolated from murine cerebral microvessels PG production was induced with either serum or epidermal growth factor (EGF). Prostaglandin H synthase (PGHS) activity peaked at 6 h after the addition of 10% serum or 50 ng/ml EGF. Increases in expression of PGHS-1 mRNA were small (7- to 10-fold) compared with PGHS-2 (30- to 120-fold), and the induction patterns were different for serum and EGF. An increase in PGHS-2 message was detected by 0.5 h of adding either agent, but peak induction occurred earlier for EGF than for serum, 1 h vs. 3 h, respectively. The response to either stimulus had returned to prestimulation levels by 12 h. The induction of PGHS-2 protein was also transient, but followed a more delayed time course (peak levels at 6 h). Induction of activity, message, and protein by either agent was blocked by 1 microM dexamethasone and attenuated by genistein (100 microM), a nonspecific tyrosine kinase inhibitor. Tyrphostin 47, a more selective EGF receptor tyrosine kinase inhibitor, dose-dependently inhibited EGF-stimulated PGHS activity, completely abolishing PG production at 100 microM. However, this inhibitor had no effect on serum-stimulated PG production. Curiously, 100 microM tyrphostin 47 enhanced EGF-induced PGHS-2 mRNA and protein expression. These data suggest that EGF induces the expression of PGHS-2 in cerebromicrovascular smooth muscle by a mechanism that requires tyrosine kinase activity and that is distinct from serum.

Development and characterization of monoclonal antibodies specific to the serotonin 5-HT2A receptor.

Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.

Serotonin receptors expressed by myelinating Schwann cells in rat sciatic nerve.

We have previously reported that Schwann cells cultured from rat sciatic nerves express 5-HT2A receptors. In this study we extend these in vitro observations to Schwann cells in situ. Since the serotonin (5-HT) levels in rat sciatic nerve are elevated following nerve injury, we examined Schwann cells in healthy and injured adult rat sciatic nerves. These nerves were double-labeled immunohistochemically with an anti-idiotypic antibody that recognizes 5-HT1B, 5-HT2A, and 5-HT2C receptors and an antibody against S100beta, a Schwann cell marker. 5-HT receptor labeling was observed in Schwann cells of healthy and regenerating nerves, but not of degenerating nerves, while S100beta labeling was observed in the Schwann cells of all nerves examined. The 5-HT receptor immunolabeling was cytoplasmic, as with the cultured Schwann cells. While staining was observed at the nodes of Ranvier, it was not restricted to these locations. These results suggest that myelinating rat Schwann cells normally express 5-HT receptors in vivo, and that receptor expression is reduced during times when 5-HT levels are elevated in the sciatic endoneurium.

The expression of serotonin receptors by cultured rat Schwann cells is a function of their differentiation: correlation with a quiescent myelinating phenotype.

Previously we reported that cultured rat Schwann cells express 5-HT2A receptors. In the present study we compared the serotonin-responsiveness of Schwann cells with their stage of differentiation. Serotonin-responsiveness occurred in cells that expressed a myelinating marker but not a nonmyelinating marker. We also examined cells to see if those responding to 5-HT were selectively proliferating or quiescent. This was accomplished by pulsing cells with bromodeoxyuridine (BrdUrd), screening them for serotonin responses, and then immunolabeling the same cells with an antibody recognizing BrdUrd. No BrdUrd+ cells responded to serotonin, whereas most BrdUrd- cells did respond, indicating that serotonin receptors are expressed in Schwann cells after they become quiescent. Moreover, detection of the responses is enhanced by reducing concentrations of mitogen-containing serum in the culture medium. Collectively, these results suggest that postmitotic Schwann cells express serotonin receptors while differentiating along the myelinating lineage.

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.

5-Hydroxytryptamine2A receptors on cultured rat Schwann cells.

Intracellular calcium responses of cultured rat Schwann cells to 5-hydroxytryptamine (5-HT) were examined using the calcium indicator dye fluo-3. Consistent changes in [Ca2+]i were observed with bath application of 5-HT and the basis of these responses was characterized. Application of 5-HT elicited a transient increase in intracellular calcium in a subpopulation of cultured Schwann cells. In many responding cells, the response recurred at approximately regular intervals following the initial transient. In some cases, these oscillations lasted for hours following removal of 5-HT from the bath. The increase in intracellular calcium evoked by 5-HT still occurred in the absence of extracellular calcium, suggesting that 5-HT induces calcium release from intracellular stores. Consistent with this hypothesis, the response to 5-HT was prevented by depletion of inositol trisphosphate-sensitive intracellular calcium stores with thapsigargin. Bath application of caffeine, known to activate Ca2+ release from ryanodine receptor-mediated stores, did not elicit an increase in [Ca2+]i. These results also suggested that 5-HT acted by stimulating a member of the 5-HT2 receptor family since this family employs inositol trisphosphate as a second messenger. In agreement with this interpretation, it was found that the 5-HT-induced intracellular calcium transients could be reversibly blocked by both ketanserin and spiperone, suggesting that the transients are mediated by 5-HT2A receptors. Additional support for this conclusion was obtained by immunocytochemistry using an anti-idiotypic antibody that recognizes a subset of 5-HT receptors.

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.