Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development: evidence for a role of these receptors in axonal growth and guidance.

There is recent evidence supporting the notion that the cannabinoid signaling system plays a modulatory role in the regulation of cell proliferation and migration, survival of neural progenitors, neuritic elongation and guidance, and synaptogenesis. This assumption is based on the fact that cannabinoid 1-type receptors (CB(1) receptors) and their ligands emerge early in brain development and are abundantly expressed in certain brain regions that play key roles in these processes. We have recently presented in vivo evidence showing that this modulatory action might be exerted through regulating the synthesis of the cell adhesion molecule L1 that is also a key element for those processes. To further explore this issue, we conducted here immunohistochemical studies aimed at determining the cellular substrates of CB(1) receptor-L1 interactions in the rat brain during late fetal development. In this period, we previously found that the activation of CB(1) receptors increased L1 synthesis in several forebrain white matter regions but not in gray matter areas. Using double labeling studies, we observed here colocalization of both proteins in fiber tracts including the corpus callosum, the adjacent subcortical white matter, the internal capsule and the anterior commissure. Experiments conducted with cultures of fetal rat cortical nerve cells revealed that L1 is present mainly in neurons but not in glial cells. This fact, together with the results obtained in the double labeling studies, would indicate that L1 and CB(1) receptors should possibly be present in axons elongating through these white matter tracts, or, alternatively, in migrating neurons. Further experiments confirmed the presence of CB(1) receptors in elongating axons, since these receptors colocalized with growth-associated protein 43 (GAP-43), a marker of growth cones, but not with synaptophysin, a marker of active synaptic terminals, in the same forebrain white matter regions. Lastly, using cultured fetal rat cortical neurons, we also observed that the activation of cannabinoid receptors increased the levels of the full-length L1 and altered those of some active proteolytic fragments of this protein whose generation has been associated with specific steps in the process of neuritic elongation in cultured neurons. In summary, we have demonstrated that the effects caused by cannabinoid agonists on L1 are facilitated by the colocalization of this cell adhesion molecule with CB(1) receptors in several forebrain white matter regions during fetal brain development. We have provided strong evidence that this phenomenon occurs in axons elongating through these white matter tracts, and we have explored in vitro how cannabinoid receptors influence L1 levels. Considering the role played by L1 in different events related to neural development, our observations support the occurrence of a physiological mechanism by which the cannabinoid system might regulate the process of axonal growth and guidance through regulating the synthesis and function of L1.

Glial expression of cannabinoid CB(2) receptors and fatty acid amide hydrolase are beta amyloid-linked events in Down's syndrome.

Recent data suggest that the endocannabinoid system (ECS) may be involved in the glial response in different types of brain injury. Both acute and chronic insults seem to trigger a shift in the pattern of expression of some elements of this system from neuronal to glial. Specifically, data obtained in human brain tissue sections from Alzheimer's disease patients showed that the expression of cannabinoid receptors of the CB(2) type is induced in activated microglial cells while fatty acid amide hydrolase (FAAH) expression is increased in reactive astrocytes. The present study was designed to determine the time-course of the shift from neuronal to glial induction in the expression of these proteins in Down's syndrome, sometimes referred to as a human model of Alzheimer-like beta-amyloid (Abeta) deposition. Here we present immunohistochemical evidence that both CB(2) receptors and FAAH enzyme are induced in Abeta plaque-associated microglia and astroglia, respectively, in Down's syndrome. These results suggest that the induction of these elements of the ECS contributes to, or is a result of, amyloid deposition and subsequent plaque formation. In addition, they confirm a striking differential pattern of distribution of FAAH and CB(2) receptors.

Cannabinoid CB2 receptors in human brain inflammation.

The presence of functional cannabinoid CB2 receptors in the CNS has provoked considerable controversy over the past few years. Formerly considered as an exclusively peripheral receptor, it is now accepted that it is also present in limited amounts and distinct locations in the brain of several animal species, including humans. Furthermore, the inducible nature of these receptors under neuroinflammatory conditions, in contrast to CB1, makes them attractive targets for the development of novel therapeutic approaches. In fact, the undesired psychoactive effects caused by CB1 activation have largely limited the clinical use of cannabinoid-related compounds that act on these receptors. In this review some recent findings on the antiinflammatory properties of CB2 receptors are presented, as well as new perspectives that have been obtained based on studies of human postmortem brain samples. In addition, various working hypotheses are also proposed and discussed.

Role of the endocannabinoid system in Alzheimer's disease: new perspectives.

The role of the endocannabinoid system in several diseases is currently under intense study. Among these, Alzheimer's disease may be a new promising area of research. We have recently reported the existence of profound changes in the location and density of several elements of this system in Alzheimer's disease tissue samples, indicating that a non-neuronal endocannabinoid system is up-regulated in activated glia. Additional data from other groups suggest that glial cells may be important elements in the regulation of endocannabinoid system activity, both in health as in disease. Some of these aspects are briefly discussed in the present review.

Association with Ets-1 causes ligand- and AF2-independent activation of nuclear receptors.

The vitamin D receptor (VDR) normally functions as a ligand-dependent transcriptional activator. Here we show that, in the presence of Ets-1, VDR stimulates the prolactin promoter in a ligand-independent manner, behaving as a constitutive activator. Mutations in the AF2 domain abolish vitamin D-dependent transactivation but do not affect constitutive activation by Ets-1. Therefore, in contrast with the actions of vitamin D, activation by Ets-1 is independent of the AF2 domain. Ets-1 also conferred a ligand-independent activation to the estrogen receptor and to peroxisome proliferator-activated receptor alpha. In addition, Ets-1 cooperated with the unliganded receptors to stimulate the activity of reporter constructs containing consensus response elements fused to the thymidine kinase promoter. There is a direct interaction of the receptors with Ets-1 which requires the DNA binding domains of both proteins. Interaction with Ets-1 induces a conformational change in VDR which can be detected by an increased resistance to proteolytic digestion. Furthermore, a retinoid X receptor-VDR heterodimer in which both receptors lack the core C-terminal AF2 domain can recruit coactivators in the presence, but not in the absence, of Ets-1. This suggests that Ets-1 induces a conformational change in the receptor which creates an active interaction surface with coactivators even in the AF2-defective mutants. These results demonstrate the existence of a novel mechanism, alternative to ligand binding, which can convert an unliganded receptor from an inactive state into a competent transcriptional activator.

Differentiation of lactotrope precursor GHFT cells in response to fibroblast growth factor-2.

The mechanisms that control the emergence of different anterior pituitary cells from a common stem cell population are largely unknown. The immortalized GHFT cells derived from targeted expression of SV40 T antigen to mouse pituitary display characteristics of somatolactotropic progenitors in that they express the transcription factor GHF-1 (Pit-1) but not growth hormone (GH) or prolactin (PRL). We searched for factors capable of inducing lactotropic differentiation of GHFT cells. PRL gene expression was not observed in cells subjected to a variety of stimuli, which induce PRL gene expression in mature lactotropes. Only fibroblast growth factor-2 (FGF-2) was able to initiate the transcription, synthesis, and release of PRL in GHFT cells. However, induction of PRL expression was incomplete in FGF-2-treated cells, suggesting that additional factors are necessary to attain high levels of PRL transcription in fully differentiated lactotropes. We also show that the FGF-2 response element is located in the proximal PRL promoter. Stimulation of PRL expression by FGF-2 requires endogenous Ets factors and these factors as well as GHF-1 are expressed at low levels in the committed precursor, suggesting that these low levels are limiting for full PRL expression. Moreover, FGF-2 effect on lactotrope differentiation is mediated, at least partially, by stimulation of the Ras-signaling pathway. Our results suggest that, indeed, GHFT cells represent a valid model for studying lactotropic differentiation and that FGF-2 could play a key role both in initiating lactotrope differentiation and maintaining PRL expression.

Synergistic activation of the prolactin promoter by vitamin D receptor and GHF-1: role of the coactivators, CREB-binding protein and steroid hormone receptor coactivator-1 (SRC-1).

PRL gene expression is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restricted manner in cells of the anterior pituitary. In pituitary GH3 cells, vitamin D increases the levels of PRL transcripts and stimulates the PRL promoter. We have analyzed the role of GHF-1 and of the vitamin D receptor (VDR) to confer vitamin D responsiveness to the PRL promoter. For this purpose we have used nonpituitary HeLa cells, which do not express GHF-1. We found that VDR activates the PRL promoter both in a ligand-dependent and -independent manner through a sequence located between positions -45/-27 in the proximal 5'-flanking region. This sequence also confers VDR and vitamin D responsiveness to a heterologous promoter. In the context of the PRL gene, VDR requires the presence of GHF-1 to activate the promoter. Truncation of the last 12 C-terminal amino acids of VDR, which contain the ligand-dependent activation function (AF2), abolishes regulation by vitamin D, suggesting that binding of coactivators to this region mediates ligand-dependent stimulation of the PRL promoter by the receptor. Indeed, expression of the coactivators, steroid hormone receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), significantly enhances the stimulatory effect of vitamin D mediated by the wild-type VDR but not by the AF2 mutant receptor. Furthermore, CBP also increases the activation of the PRL promoter by GHF-1 and the ligand-independent activation by both wild-type and mutant VDR.

Activation of the prolactin gene by peroxisome proliferator-activated receptor-alpha appears to be DNA binding-independent.

Although the effects of the peroxisome proliferator-activated receptors (PPARs) have been studied primarily in adipocytes and liver, the wide distribution of these receptors suggests that they might also play a role in other cell types. We present evidence that PPAR activators stimulate the expression of the prolactin gene in pituitary GH4C1 cells. Transfection assays in non-pituitary HeLa cells showed that stimulation of the prolactin promoter by PPARalpha requires the presence of the transcription factor GHF-1 (or Pit-1). Proximal promoter sequences confer responsiveness to PPARalpha, and activation by this receptor is lost concomitantly with the response to GHF-1. Surprisingly, expression of the retinoid X receptor (RXR) abolishes stimulation by PPARalpha. Furthermore, the promoter region that confers PPARalpha responsiveness does not contain a PPAR response element. This suggests that the transcriptional effect of PPARalpha might be mediated by protein-protein interactions rather than by binding of PPAR/RXR to the promoter. A direct interaction between PPARalpha and GHF-1 was confirmed by in vitro binding studies. Expression of the coactivators SRC-1 and CREB-binding protein, which bind to PPAR, also enhanced the responsiveness of the prolactin promoter to PPARalpha. Furthermore, CREB-binding protein also significantly increased activation by GHF-1, and both proteins associated in vitro. Thus, PPARalpha, a receptor that normally acts as a ligand-dependent transcription factor by binding to specific DNA sequences in one context, can also stimulate the prolactin promoter by association with GHF-1 and coactivator proteins.

Insulin-like growth factor-1 stimulates rat prolactin gene expression by a Ras, ETS and phosphatidylinositol 3-kinase dependent mechanism.

We have examined the influence of insulin-like growth factor I (IGF-1) on prolactin gene expression in rat pituitary GH4C1 cells. Incubation with IGF-1 increases prolactin mRNA levels and activates the prolactin promoter in transient transfection assays. A similar degree of activation is observed with constructs extending to -3000 and -176 base pairs of the prolactin 5' flanking region, indicating that the IGF-1 response element is located in the proximal promoter sequences. A plasmid containing 101 base pairs shows a partial stimulation by IGF-1, and the response is lost in a deletion to -76 base pairs. The Ras oncoproteins have been implicated as a critical signaling component in mediating the effect of growth factor receptor tyrosine kinases. Expression of oncogenic RasVal12 mimics the effect of IGF-1 on the prolactin promoter, and a dominant negative Ras, RasAsn17, blocks IGF-1-mediated stimulation. Dominant negative mitogen-activated protein kinase (MAPK) also reduces the activation of the prolactin promoter by IGF-1. Ets transcription factors have been described to lie downstream of Ras and MAPK in the signaling pathway leading to prolactin gene activation. Mutation of two Ets binding sites in the promoter region between -101 and -76 abolishes the response to IGF-1. Furthermore, a dominant negative Ets vector strongly reduces the response of the prolactin promoter to IGF-1 and Ras. The endogenous concentration of Ets-related proteins is not limiting in GH4C1 cells for the IGF-1 effect. However, c-Ets-1 and GHF-1 act synergistically in HeLa cells with the IGF-1 receptor, reconstituting pituitary IGF-1 responsiveness. The response to IGF-1 in GH4C1 cells is still observed after transfection with RasVal12 suggesting that, although Ras is required, IGF-1 could stimulate other pathway/s in addition to Ras. Wortmanin, an inhibitor of phosphatidylinositol-3 kinase (PI-3 kinase), also prevents the response of the prolactin promoter to IGF-1. These results show that both the Ras/MAPK/Ets pathway, as well as the activation of PI-3 kinase are involved in the signaling mechanism leading to prolactin expression by IGF-1 in GH4C1 cells.

Growth hormone induces somatostatin and insulin-like growth factor I gene expression in the cerebral hemispheres of aging rats.

The neuropeptide somatostatin (SS) plays a role as a modulator of cognitive functions and as a potential tropic factor in the central nervous system. A reduction in SS levels has been demonstrated in the aging brain and in dementia. In addition, insulin-like growth factor I (IGF-I) acts as a paracrine factor in multiple GH actions and is also found in the cerebral hemispheres, where it exerts neurotropic effects. We used aging rats as an in vivo model of GH deficiency to study the possible participation of exogenous GH in the modulation of the cerebral hemispheric SS and IGF-I. Two sets of experiments were carried out. In the first set, the age-related patterns of GH, IGF-I, and SS in the serum, pituitary, and cerebral hemispheres were established. In the second experimental set, 90-day-old (adult) and 2-yr-old (aging) male rats received recombinant human GH (200 micrograms/ sc-day) or vehicle for 7 consecutive days. The serum levels of rat GH and IGF-I as well as pituitary GH messenger RNA decreased in 2-yr-old rats compared with those in adult rats. After GH treatment, pituitary GH messenger RNA levels decreased markedly in the 90-day-old and 2-yr-old rats. Serum immunoreactive GH decreased in the adult animals, whereas it remained unaffected in the aging ones, whereas serum IGF-I levels were not altered by GH treatment in either group. Immunoreactive levels and messenger RNA of both SS and IGF-I were low in the cerebral hemispheres of aging rats, but were restored to the levels found in adult rats after GH treatment. As treatment did not induce changes in the serum IGF-I levels, these results provide evidence of a stimulatory action of peripherally administered GH on the regulation of SS and IGF-I genes in the aging rat in the central nervous system. These data also show a new target action for GH and could provide a molecular basis for the improvement of some symptoms of GH deficiency that occurs after recombinant human GH treatment.

Regulation of somatostatin gene expression by veratridine-induced depolarization in cultured fetal cerebrocortical cells.

The stimulatory effect of veratridine (VTD) depolarization upon somatostatin mRNA (SS mRNA) levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 100 microM VTD exposure for 30 min, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media without affecting SS mRNA levels. These levels increased when exposure to depolarization stimuli was prolonged up to 3 or more hours. At this time, veratridine acted as a secretagogue, stimulating somatostatin secretion, but was also effective in stimulating somatostatin mRNA levels. These changes were blunted by the Na+ channel blockade tetrodotoxin (TTX), and partially abolished by the Ca2+ channel antagonist, verapamil (VPM). To study whether VTD may affect mRNA stability we determine the rate of disappearance of SS mRNA after inhibition of transcription by actinomycin D and demonstrated that VTD stimulation did not stabilize the SS mRNA. These results indicate that the induction of SS mRNA expression by VTD involves the modulation of Ca2+ and Na+ channels. The time course study confirmed that the VTD-induced SS mRNA accumulation is time-dependent, and requires a prolonged exposure to stimulate SS gene expression. VTD stimulation does not modify the SS mRNA rate of degradation.

Triiodothyronine regulates somatostatin gene expression in cultured fetal rat cerebrocortical cells.

The effect of triiodothyronine (T3) on somatostatin (SS) mRNA levels in cultured fetal rat cerebrocortical cells was studied. Two different experimental approaches were sought. They differed in the length of time in which cells were deprived of thyroid hormones prior to the addition of exogenous T3. When the cells were not deprived of thyroid hormones, T3 caused a dose-related decrease in SS mRNA content at all doses tested. However, when the cells were deprived of T3 for 24 h, a biphasic effect was observed. These findings suggest that T3 regulates SS gene expression in fetal cultured cerebrocortical cells.

Induction of vasoactive intestinal peptide gene expression and prolactin secretion by insulin-like growth factor I in rat pituitary cells: evidence for an autoparacrine regulatory system.

The effects of recombinant human insulin-like growth factor I (IGF-I) on both vasoactive intestinal peptide (VIP) and PRL production and gene expression were studied using rat anterior pituitary cell cultures grown in serum-free defined medium. We also examined whether pituitary VIP could be involved in the PRL response to IGF-I and hence in a paracrine regulatory system. Exposure of cultured anterior pituitary cells to IGF-I (2.6 nM) for 3 h caused a significant decrease in both VIP content and media PRL. Treatment with IGF-I (from 0.65-5.2 nM) for 48 h increased VIP production and VIP messenger RNA (mRNA) accumulation, whereas only an increase in media and intracellular PRL content without changes in mRNA was observed. In all these experiments, IGF-I led to a decrease in both GH secretion and expression. Immunoglobulins G purified from VIP antiserum inhibited IGF-I-induced PRL release without affecting intracellular and mRNA levels. The inhibition of both GH secretion and gene expression induced by IGF-I was not blocked by VIP antiserum. In conclusion, these results indicate that IGF-I induces VIP gene expression, and its secretion and also increases PRL secretion. The effect of IGF-I on PRL release is specifically mediated by VIP through a paracrine or autocrine mechanism.

Effect of potassium-induced depolarization on somatostatin gene expression in cultured fetal rat cerebrocortical cells.

The stimulatory effect of potassium depolarization upon somatostatin (SS) mRNA levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 56 mM K+ exposure for 30 min, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media and decreased SS mRNA levels. These were increased when exposure to depolarization stimuli was prolonged up to 3 or more hr. At this time, potassium (30 and 56 mM) acted as a secretagogue, stimulating SS secretion, but was also effective in stimulating SS mRNA levels, suggesting that SS secretion can be coupled to SS mRNA accumulation. These changes were inhibited by the Ca2+ channel antagonist verapamil. In contrast, Na+ channel blockade by TTX did not modify the 24 hr potassium-induced increase in SS mRNA, although it partially abolished potassium-induced SS secretion. Examination of the rate of disappearance of SS mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that K+ stimulation of cerebrocortical cells stabilized the SS mRNA. These results suggest that the induction of SS mRNA expression by K+ is dose dependent, and involves the modulation of ion channels. The time-course study confirmed that the K(+)-induced SS mRNA accumulation is time dependent, chronic activation of the Ca2+ channels being necessary to stimulate SS gene expression. K+ stimulation may also increase the level of SS mRNA in cerebrocortical cells by reducing its rate of degradation.

Neurotransmitter regulation of somatostatin secretion by fetal rat cerebral cortical cells in culture.

Extensive studies exploring the regulation of hypothalamic somatostatin GHRIH release have been reported, but the factors regulating GHRIH release in the cerebral cortex have not been well defined. We have studied the effects of central neurotransmitters on GHRIH secretion by cultured fetal rat cerebral cortical cells and on intracellular GHRIH levels. Cells maintained in vitro for 15-20 days were incubated with dopamine (DA), acetylcholine (ACh), gamma-aminobutyric acid (GABA), norepinephrine (NE), serotonin (SE) or histamine (His) (10(-11) M to 10(3) M) for 30 minutes. Following incubation, immunoreactive GHRIH was measured by RIA in cell extracts and incubation media. DA increased intracellular GHRIH content but have no effect on GHRIH in the media. Both media and intracellular GHRIH content were significantly reduced by GABA and SE. The effect of NE was stimulatory at low (10(-9) M) and inhibitory at high (10(-5) M to 10(-3) M) concentrations. ACh was found to increase media GHRIH and to decrease intracellular GHRIH content; 30 min exposure to His did not significantly modify either media or intracellular GHRH. Our findings with fetal rat cerebral cortical cells in culture demonstrate that endogenous neurotransmitters do have the capacity to directly influence GHRIH regulation.

Potassium-induced depolarization stimulates somatostatin gene expression in cultured fetal rat cerebrocortical cells.

The stimulatory effect of potassium depolarization upon somatostatin mRNA (SS mRNA) levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 56 mM K+ concentration for 24 hours, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media and SS mRNA levels, suggesting that somatostatin secretion can be coupled to SS mRNA accumulation. These changes were inhibited by the Ca2+ channel antagonist verapamil (VPM). In contrast, Na+ channel blockade by tetrodotoxin (TTX) did not modify the 24 h potassium-induced increase in SS mRNA. These results suggest that the induction of SS mRNA expression by K+ involves the modulation of calcium ion channels.

Regulation of somatostatin and growth hormone-releasing factor by gonadal steroids in fetal rat hypothalamic cells in culture.

The mechanism underlying the sexually dimorphic pattern of growth hormone (GH) secretion in the rat has not been clearly elucidated. In the present study, we assayed the possible direct effect of gonadal steroids on both somatostatin (SS) and growth hormone-releasing factor (GRF) in fetal rat hypothalamic cells in culture. Hypothalamic cells, obtained by mechanical dispersion, were maintained as monolayer cultures in serum-supplemented medium. After 20 days in culture, cells were incubated with serum free medium containing testosterone (T, 10, 20, 40 ng/dl) or estradiol (E, 0.1, 1, 10 ng/dl) for 48 h. At the end of the experiments, immunoreactive SS (IR-SS) and immunoreactive GRF (IR-GRF) were measured by specific radioimmunoassays (RIAs) in media and cell extracts. After 48 h of incubation with testosterone, somatostatin in both media and cells was significantly reduced. On the contrary, this treatment lead to a dose-dependent increase in media and cell GRF content. When cells were incubated with estradiol for 48 h, a significant inhibition in medium SS release was observed, whereas intracellular SS slightly increased at the highest concentration of 10 ng/dl. Estradiol treatment resulted in an inconsistent decrease in media and cells IR-GRF. Our results indicate that both SS and GRF are under the influence of testosterone and estradiol acting at the hypothalamic level, and furthermore suggest that at this stage of brain development, gonadal steroids may regulate GH secretion through their ability to modulate hypothalamic SS and GRF.

Role of locally produced growth hormone-releasing factor in somatostatin regulation by fetal rat brain cells in culture.

To determine the possible physiological role of endogenous growth hormone-releasing factor (GRF) in the neuronal content and release of cerebral somatostatin (SS), we studied the effect of endogenous GRF blockade on the immunoreactive SS (IR-SS) content of cells and media in fetal rat cerebral cortical and hypothalamic cells in culture. Cells were cultured in minimum essential medium (MEM) with 10% fetal calf serum and 10% horse serum. After 7-10 days in vitro, media were replaced with MEM without sera containing anti-GRF immunoglobulins G (IgG) for 1, 5 or 24 h. Controls were incubated with equal amounts of IgG from normal rabbit serum (NRS). In another group of experiments, cells were incubated with GRF (10(-11) to 10(-7) M) for 1 or 24 h. Long-term exposure (24 h) to anti-GRF IgG resulted in decreased media and intracellular IR-SS content, in both cerebral cortical and hypothalamic cells. 24 h treatment with GRF caused a dose-dependent increase in the IR-SS content of cells and media, the stimulatory action being abolished by the addition of anti-GRF to plates containing 10(-7) M GRF. On the contrary, when cells were exposed to anti-GRF IgG for 1 h, IR-SS increased in the media as compared to the control group. Short-term incubation (1 h) with GRF (10(-9) to 10(-7) M) resulted in a dose-dependent inhibition of IR-SS content in the cells and media. This inhibitory action was partially prevented by the addition of anti-GRF to plates containing 10(-7) M GRF.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of platelets with subendothelium in rats treated with PCA-4230, a new antithrombotic agent. Effect of PCA-4230 on experimental thrombosis.

The effects of a new antithrombotic compound, PCA-4230, versus ticlopidine were investigated using an experimental thrombosis and vascular endothelial injury model in rats. Both PCA-4230 and ticlopidine protected rat arteries from the formation of prominent thrombi and most of microthrombi without modifying the formation of a first platelet monolayer. Neither coagulation parameters nor fibrinolysis were modified by these antithrombotic drugs. Neither PCA-4230 nor ticlopidine affected thromboxane A2 production in rats, whereas unlike PCA-4230, ticlopidine inhibited ex vivo fibrinogen binding to the fibrinogen receptor found on the platelet membrane. In conclusion, PCA-4230 and ticlopidine inhibited thrombus formation in vivo by a platelet-dependent mechanism which may be different for one or the other drug in spite of the fact that the protective effect measured in this thrombosis model is quite similar for either PCA-4230 or ticlopidine. The above-mentioned results clearly show that PCA-4230 is a new potent agent with both antivascular-damaging and antiplatelet activities, and devoid of effects on coagulation and fibrinolytic systems.

Thyroid hormones regulate release and content of vasoactive intestinal peptide in cultured fetal cerebral cortical cells.

The effects of thyroid hormones (TH) on brain immunoreactive-vasoactive intestinal peptide (IR-VIP) secretion and content in cultured fetal rat cortical cells were studied. Cerebral cortical cells were maintained as monolayer cultures for 14-18 days. T3 or T4 (10(-7) M) caused a time-dependent decrease in total IR-VIP. Significant suppression was observed following treatment periods of 6 h or longer (24 and 48 h). Depending on the length of time cells had been deprived of TH prior to the addition of exogenous T3 or T4, these two thyroid hormones had different effects on IR-VIP accumulation. Both T3 and T4 caused a dose-dependent suppression or IR-VIP accumulation when there was no deprivation period or when it lasted 4 h. However, a biphasic effect was observed when cells were deprived of TH for 17 and 24 h: low doses of T3 or T4 (from 10(-12) to 10(-10) M) significantly increased (p less than 0.05) total IR-VIP, while high T3 or T4 doses (10(-8) and 10(-7) M) caused a significant decrease (p less than 0.01). The TH action was furthermore shown to be reversible. After T3 (10(-7) M) removal and subsequent incubation in serum-free medium for 6, 24 and 48 h, T3-treated and control cells exhibited similar levels of IR-VIP release and content. At this time, a new exposure to T3 (10(-7) M) again had a suppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)