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At least 307 records · Page 17Linked to original sources
PubMed2002

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor.

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.

Animals↗
PubMed2000

The role of cytoplasmic dynein in the human brain developmental disease lissencephaly.

Lissencephaly is a brain developmental disorder characterized by disorganization of the cortical regions resulting from defects in neuronal migration. Recent evidence has implicated the human LIS-1 gene in Miller-Dieker lissencephaly and isolated lissencephaly sequence. LIS-1 is homologous to the fungal genes NudF and PAC1, which are involved in cytoplasmic dynein mediated nuclear transport, but it is also almost identical to a subunit of PAF acetylhydrolase, an enzyme which inactivates the lipid mediator platelet activating factor. Recent evidence from our laboratory has revealed that cytoplasmic dynein coimmunoprecipitates with LIS-1 in bovine brain cytosol, supporting a role in the dynein pathway in vertebrates. Overexpression of LIS-1 interferes with cell division, with noteworthy effects on chromosome attachment to the mitotic spindle and on the interaction of astral microtubules with the cell cortex. Other aspects of dynein function, such as the organization of the Golgi apparatus, are not affected. Together, these results suggest a role for LIS-1 in cytoplasmic dynein functions involving microtubule plus-ends. Furthermore, they suggest that mutations in LIS-1 may produce a lissencephalic phenotype either by interfering with the movement of neuronal nuclei within extending processes, or by interference with the division cycle of neuronal progenitor cells in the ventricular and subventricular zones of the developing nervous system.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2001

PACAP activates PKA, PKC and Ca(2+) signaling cascades in rat neuroepithelial cells.

Several studies have reported that the PAC(1) receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca(2+) concentration ([Ca(2+)](i)) was increased by PACAP, but not VIP, in a concentration range from 10(-12) to 10(-8) M via the PAC(1)-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC(1)-R is linked to [Ca(2+)](i) signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.

Animals↗
PubMed1999

Insulinotropin PACAP potentiates insulin-stimulated glucose uptake in 3T3 L1 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized in pancreatic nerve fibers and islets and potently augments glucose-induced insulin secretion. The present study explored a possible extra-pancreatic action of PACAP. The specific PACAP receptor (PAC1 receptor) was expressed in the rat fat tissue and 3T3-LI adipocytes. PACAP-38 (10 nM) significantly enhanced insulin-induced 2-deoxyglucose uptake by 3T3-L1 adipocytes. Insulin-stimulated phosphatidylinositol 3-kinase activity was further increased by PACAP-38, whereas the tyrosine-phosphorylation of insulin receptor beta-subunit and insulin receptor substrate-1 was unaltered by PACAP-38. These results reveal that PACAP-38 enhances insulin-induced glucose uptake, an effect probably mediated by insulin-stimulated phosphatidyl-inositol 3-kinase, and that PACAP potentiates not only insulin secretion, but also insulin action in adipocytes.

3T3 Cells↗
PubMed2003

Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide inhibit feeding in the chick brain by different mechanisms.

Intracerebroventricular (ICV) injections of pituitary adenylate cyclase-activating polypeptide-38 (PACAP) and vasoactive intestinal peptide (VIP) inhibit feeding in chicks. However, the precise anorexigenic mechanisms have not been investigated, since both peptides activate the VPAC receptor in mammals. We investigated which receptor mediates the anorexigenic effects of these peptides in chicks. ICV co-injection of PACAP (6-38), a PAC1 receptor antagonist, attenuated the anorexigenic effect of PACAP but not VIP. On the other hand, ICV co-injection of [D-p-Cl-Phe6, Leu17]-VIP, a VPAC receptor antagonist, did not affect the effects of both peptides. Although these results imply that the effect of VIP was not specific, a subsequent experiment demonstrated that ICV injection of anti-chicken VIP antiserum stimulated feeding and suggested that endogenous VIP inhibits feeding in the chick brain. Collectively, the data suggest that the anorexigenic mechanism of PACAP is different from that of VIP and that an undiscovered VIP receptor may be present in the chicken brain.

Animals↗
PubMed1999

Pituitary adenylate cyclase-activating polypeptide receptors during development: expression in the rat embryo at primitive streak stage.

The distribution and localization of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor the PAC1 receptor (previously called the type 1 PACAP receptor or PVR1), which binds PACAP, but not vasoactive intestinal peptide, with high affinity] were first investigated in rats with in situ hybridization for its messenger RNA, and with immunohistochemical methods during prenatal and postnatal development. The expression of PACAP receptor messenger RNA was first detected in the rat embryo at the primitive streak stage as early as embryonic day 9, and it was intensely expressed in the neural plate. PACAP receptor messenger RNA was also intensely expressed in the neuroepithelia of the mesencephalon and rhombencephalon at embryonic day 11, and expressed in the basal telencephalon, hippocampal formation neuroepithelium, cortical neuroepithelium and cerebellar neuroepithelium after embryonic day 13. It was also expressed in the olfactory bulb neuroepithelium after embryonic day 16, and in mature regions of the older embryos. In postnatal developing brains, PACAP receptor messenger RNA was intensely expressed in the olfactory bulb, hippocampal formation, cerebellum and other scattered regions. The localization of PACAP receptor-like immunoreactivity coincided well with that of the gene transcripts. We also used reverse transcription-polymerase chain reaction methods to determine the expression of the splice variants of the PACAP receptor gene. At each ontogenetic stage of the rat from embryonic day 9 to postnatal day 60, two major products were detected with reverse transcription-polymerase chain reaction, a thick band (303 base pairs) corresponding to the short splice variant of the receptor that lacks both the "hip" and "hop" cassettes, and a thin band (387 base pairs) corresponding to the splice variant that contains one cassette of "hop" or "hip". There was no evidence for the other larger splice variants. Some of the amplified products were sequenced and found to have the exact sequences of "PACAP receptor" and "PACAP receptor-hopl", which are coupled to different signal transduction pathways. These results indicate that the PACAP receptor is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development, and that the major splice variants are "PACAP receptor" and "PACAP receptor-hopl". The initial mapping of ontogenetic localization of the PACAP receptor provides the basis for a better understanding of the functions of PACAP and its receptors during the development of the brain.

Animals↗
PubMed2001

Neuroendocrine differentiation of the LNCaP prostate cancer cell line maintains the expression and function of VIP and PACAP receptors.

The molecular mechanisms involved in differentiation of prostate cancer cells to a neuroendocrine (NE) cell phenotype are not well understood. Here we used the androgen-dependent human prostate cancer cell line LNCaP to perform a systematic and broad analysis of the expression, pharmacology, and functionality of vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptors. Reverse transcription polymerase chain reaction experiments, together with pharmacological approaches with a set of specific agonists and antagonists, demonstrated the presence of the three VIP/PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 with a major role for VPAC1, acting through adenylate cyclase (AC) stimulation. An essentially similar pattern was observed by NE differentiated cells (4 days after serum deprivation) in spite of the important morphological changes observed. However, the expression of the prostate-specific antigen (PSA) decreased in NE cells (and increased again by dihydrotestosterone, DHT, treatment). The present demonstration of the induction of NE transdifferentiation in LNCaP cells by increasing concentrations of VIP adds value to previous observations on the role of cAMP in this process, an interesting topic in the comprehension of the molecular changes that are involved in the progression of prostate cancer to androgen independence.

Adenylyl Cyclases↗
PubMed2004

Effect of Fgd1 on cortactin in Arp2/3 complex-mediated actin assembly.

Mutations in faciogenital dysplasia protein (Fgd1) result in the human disease faciogenital dysplasia (FGDY). Fgd1 contains a RhoGEF domain specific for Cdc42. Fgd1 also contains a Src homology (SH3) binding domain (SH3-BD) that binds directly to the SH3 domain of cortactin, which promotes actin assembly by actin-related protein (Arp)2/3 complex. Here, we report the effect of ligation of cortactin's SH3 domain by the Fgd1 SH3-BD on actin polymerization in vitro. Glutathione S-transferase (GST)-fused Fgd1 SH3-BD enhanced the ability of cortactin to stimulate Arp2/3-mediated actin polymerization. However, a synthetic peptide containing only the SH3-BD sequence had no effect. The SH3-BD peptide bound to cortactin and inhibited the effect of GST-Fgd1 SH3-BD, suggesting that GST dimerization was responsible for the stimulating effect of GST-Fgd1 SH3-BD. When GST-Fgd1 SH3-BD was prepared as a heterodimer with a control GST fusion protein (GST-Pac1), no stimulatory effect on actin polymerization was observed. In addition, when cortactin was dimerized via its N-terminus, away from the C-terminal SH3 domain, actin polymerization with Arp2/3 complex increased markedly, compared to free cortactin. Thus, cortactin ligated by Fgd1 is fully active, indicating that the cell can use Fgd1 to target actin assembly. Moreover, if Fgd1 is multimerized, then cortactin's activity should be enhanced. Fgd1 and cortactin may participate as scaffolds and signal transducers in a positive feedback cycle to promote actin assembly at the cell cortex.

Actin-Related Protein 2↗
PubMed2005

Parallels in rRNA processing: conserved features in the processing of the internal transcribed spacer 1 in the pre-rRNA from Schizosaccharomyces pombe.

Despite the large differences in their length and nucleotide composition, comparative analyses of the internal transcribed spacer 1 (ITS1) of widely divergent eukaryotes have suggested a simple core structure consisting of a central extended hairpin and lesser hairpin structures at the maturing junctions [Lalev, A. I., and Nazar, R. N. (1998) J. Mol. Biol. 284, 1341-1351]. In this study, the ITS1 in the pre-rRNA transcripts of Schizosaccharomyces pombe cells was examined with respect to structural features that underlie rRNA maturation. When plasmid-associated rRNA genes were expressed in vivo, a deletion of any major hairpin structure significantly reduced or eliminated both small and large subunit RNAs. Only changes in the central extended hairpin or junction regions, however, entirely eliminated plasmid-derived RNAs or resulted in elevated precursor levels. Structure-disrupting base substitutions within the RAC protein complex binding site in the extended hairpin indicated that the secondary structure was critical for rRNA maturation; composition or other changes with respect to the binding site had only modest effects. A similar disruption at the junction with the 18S rRNA also had striking effects on rRNA maturation, including a highly elevated level of unprocessed precursor and a surprisingly critical effect on 5.8S rRNA production. As previously observed with the 3' external transcribed spacer, the results are consistent with a maturation mechanism in which an initial cleavage in the 5' junction region may be directed by the RAC protein complex. Although not critical to rRNA processing, analyses of termini based on S1 nuclease protection as well as cleavage studies, in vitro, with Pac1 ribonuclease raise the possibility that in eukaryotes, as previously observed in bacteria, the RNase III homologues normally initiate the separation of the subunit RNAs.

Binding Sites↗
PubMed1997

A novel human ERK phosphatase regulates H-ras and v-raf signal transduction.

A cDNA encoding a novel human extracellularly-regulated kinase (ERK) phosphatase, designated B59, was isolated from a B5/589 human mammary epithelial cell cDNA library. The 1104 nucleotide open reading frame encodes 368 amino acids including the highly conserved catalytic site sequence of protein phosphotyrosine phosphatases (PTPs), VXVHCXXGXXR, at amino acid position 276-287. The predicted 70 amino acid stretch surrounding the HC motif shares significant sequence identity with other human dual specificity PTPs (dsPTPs), including the known ERK PTPs CL100, PAC1, B23, as well as the dsPTPs VH-1 and VHR. B59 protein synthesized in vitro in a rabbit reticulocyte lysate dephosphorylated rat ERK1 and ERK2 proteins whose phosphorylation had been stimulated by v-mos kinase added to the lysate. Ectopic expression of B59 in NIH3T3 fibroblasts inhibited the induction of an oncogene-responsive promoter by the dominant-activating raf mutant, raf-BXB. Moreover, cotransfection of NIH3T3 cells with B59 inhibited morphological transformation by H-ras and v-raf oncogenes. These results suggest that B59 suppresses the transforming activity of H-ras or v-raf oncogenes through ERK dephosphorylation and inactivation.

3T3 Cells↗
PubMed2001

Green fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

Animals↗
PubMed2004

Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail.

Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the alpha and beta tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the alpha tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membrane-distal region of the alphaIIb tail. Alanine substitution of the RPP residues in the alphaIIb tail rendered alphaIIbbeta3 constitutively active in a metabolic energy-dependent manner. Although an alphaIIb/alpha6Abeta3 chimaeric integrin, in which the alphaIIb tail was replaced by the alpha6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the alpha6A tail inhibited binding of an activation-dependent antibody PAC1. In alphaIIb/alpha6Abeta3, deleting the TSDA sequence from the alpha6A tail or single amino acid substitutions of the TSDA residues inhibited alphaIIb/alpha6Abeta3 activation and replacing the membrane-distal region of the alphaIIb tail with TSDA rendered alphaIIbbeta3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the alphaIIb tail containing the RPP sequence of the membrane-distal region failed to activate alphaIIbbeta3. These results suggest that the RPP sequence after the GFFKR motif of the alphaIIb tail suppresses energy-dependent alphaIIbbeta3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by alpha subunit tails.

Amino Acid Sequence↗
PubMed2003

An essential role for peptidergic signalling in the control of circadian rhythms in the suprachiasmatic nuclei.

Two structurally related neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP), colocalized with glutamate in neurones of the retinohypothalamic tract, and vasoactive intestinal peptide (VIP), present in light-responsive cells of the suprachiasmatic nuclei (SCN), appear to play distinct and important roles in the control of mammalian circadian rhythms. Mice deficient in the PACAP-selective PAC1 receptor exhibit altered responsiveness of the SCN clock to light-induced phase-shifts, but display robust circadian patterns of wheel-running behaviour. By contrast, our studies of mice lacking the VPAC2 receptor, which responds to both PACAP and VIP, indicate that this receptor plays a critical role in rhythm generation in the SCN. The predominant factor determining wheel-running activity in VPAC2 receptor null (Vipr2-/-) mice is "masking" by light. Mutant animals re-entrain immediately to advances or delays in the light/dark cycle and do not exhibit robust circadian rhythms of behaviour when in constant darkness. The mice do not exhibit circadian expression of core clock genes (mPer1, mPer2, mCry1), or of the clock-controlled gene arginine vasopressin (AVP), in the SCN. We propose that VIP signalling between SCN neurones provides a paracrine reinforcing signal that is essential for sustained rhythm generation. The presence of VIP signalling in the SCN may explain why SCN neurones are capable of generating long-lasting self-sustained oscillations, whereas rhythmic clock gene expression in other tissues is dependent on periodic reinforcement by neural or hormonal signals.

Animals↗
PubMed1999

Long-term enhancement of REM sleep by the pituitary adenylyl cyclase-activating polypeptide (PACAP) in the pontine reticular formation of the rat.

In rats, rapid eye movement (REM) sleep can be elicited by microinjection of vasoactive intestinal polypeptide (VIP) into the oral pontine reticular nucleus (PnO). In the present study, we investigated whether this area could also be a REM-promoting target for a peptide closely related to VIP: the pituitary adenylyl cyclase-activating polypeptide (PACAP). When administered into the posterior part of the PnO, but not in nearby areas, of freely moving chronically implanted rats, PACAP-27 and PACAP-38 (0.3 and 3 pmol) induced a marked enhancement (60-85% over baseline) of REM sleep for 8 h that could be prevented by prior infusion of the antagonist PACAP-(6-27) (3 pmol) into the same site. Moreover, injections of PACAP into the centre of the posterior PnO resulted in REM sleep enhancement which could last for up to 11 consecutive days. Quantitative autoradiography using [125I]PACAP-27 revealed the presence in the PnO of specific binding sites with high affinity for PACAP-27 and PACAP-38 (IC50 = 2.4 and 3.2 nM, respectively), but very low affinity for VIP (IC50 > 1 microM). These data suggest that PACAP within the PnO may play a key role in REM sleep regulation, and provide evidence for long-term (several days) mechanisms involved in such a control. PAC1 receptors which have a much higher affinity for PACAP than for VIP might mediate this long-term action of PACAP on REM sleep.

Animals↗
PubMed2000

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation.

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole-cell patch-clamp recording techniques. VIP and PACAP (</= 10 nM) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr1, D-Phe2]-GRF 1-29, amide (100 nM), a selective antagonist of VPAC1 and VPAC2 receptors; whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nM PACAP6-38, a PAC1 and VPAC2 receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 microM GTPgammaS or GDPbetaS inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTPgammaS alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alphao, alphai-1,2, alphai-3 or beta G-protein subunits. Only the anti-Galphao and anti-Gbeta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive Go protein.

Acetylcholine↗
PubMed2003

Light-induced phase shift in the Syrian hamster (Mesocricetus auratus) is attenuated by the PACAP receptor antagonist PACAP6-38 or PACAP immunoneutralization.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.

Animals↗
PubMed2002

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death.

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

Animals↗
PubMed1996

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Animals↗