C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4.

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a strong inhibitor of cell proliferation. We found that C/EBPalpha directly interacts with cdk2 and cdk4 and arrests cell proliferation by inhibiting these kinases. We mapped a short growth inhibitory region of C/EBPalpha between amino acids 175 and 187. This portion of C/EBPalpha is responsible for direct inhibition of cyclin-dependent kinases and causes growth arrest in cultured cells. C/EBPalpha inhibits cdk2 activity by blocking the association of cdk2 with cyclins. Importantly, the activities of cdk4 and cdk2 are increased in C/EBPalpha knockout livers, leading to increased proliferation. Our data demonstrate that the liver-specific transcription factor C/EBPalpha brings about growth arrest through direct inhibition of cdk2 and cdk4.

Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation.

BACKGROUND: Substantial evidence implicates the neuropeptide substance P (SP) in mucosal immunoinflammatory responses. Autoradiographic studies have suggested a disturbance in SP receptor expression in inflammatory bowel disease (IBD). AIMS: Because of technical limitations such as poor cellular resolution with autoradiography, we used molecular methods to specifically localise the cellular expression of the neurokinin-1 receptor (NK-1R) in IBD colon, and to quantitate NK-1R mRNA expression levels therein. METHODS: In situ hybridisation and immunohistochemistry were used to localise NK-1R mRNA and protein, respectively, in normal, ulcerative colitis (UC), and Crohn's disease (CD) colonic resections. NK-1R mRNA expression levels of normal, UC, and CD mucosal biopsies were quantitated by competitive reverse transcription-polymerase chain reaction. RESULTS: NK-1R expression was localised to lamina propria mononuclear cells, epithelium, submucosal vasculature, smooth muscle, and myenteric plexus of normal and IBD colon. No ectopic NK-1R expression was observed in IBD. However, we found increased numbers of NK-1R expressing lymphoid cells in IBD tissue, aberrant negative epithelial expression of NK-1R in UC, and increased expression of NK-1R in CD myenteric plexus. Quantitation of NK-1R mRNA expression in IBD colonic mucosal biopsies revealed marked upregulation of NK-1R mRNA levels compared with non-inflamed mucosal expression levels (p<0.01). CONCLUSIONS: This report demonstrates the strategic localisation and upregulation of NK-1R expression in IBD colon, and thereby suggests the involvement of substance P in the pathophysiological symptoms of IBD.

Differential expression of neurokinin-1 receptor by human mucosal and peripheral lymphoid cells.

Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

Substance P (neurokinin-1) receptor is a marker of human mucosal but not peripheral mononuclear cells: molecular quantitation and localization.

Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.

An examination of the relationship between mu-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins.

The hypothesis that mu-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than mu-opioid agonists having higher antinociceptive efficacy was tested. Supraspinal antinociceptive efficacy for the three mu-opioid agonists morphine, [D-Ala2, NMePhe4, Gly5-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 degrees C warm-water tail-flick test was evaluated 18-24 h after intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA). The beta-FNA pretreatment (0.2-2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1-3.0 micrograms) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious mu agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory Gi-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious mu agonists have greater capacity to utilize CTX-sensitive stimulatory Gs-proteins than do mu-agonists with lower efficacy.

A Gi2alpha antisense oligonucleotide differentiates morphine antinociception, constipation and acute dependence in mice.

In the same mice in which the intracerebroventricular (i.c.v.) administration of antisense oligodeoxyribonucleotide (oligo) directed against the Gi2alpha (but not Gi1alpha, Gi3alpha or G(s)alpha) G-protein subunits attenuated i.c.v. morphine-induced antinociception in the tail-flick test, none of the oligos altered naloxone-precipitated jumping (acute dependence). Likewise, none of the oligos significantly altered morphine-induced constipation. Hence, i.c.v. morphine-induced antinociception might be preferentially mediated via transduction pathway(s) different from constipation or acute dependence, offering novel opportunities for drug discovery.

Central administration of p-octopamine to mice: assessment of antinociception.

Administration of p-octopamine by intracerebroventricular (i.c.v.) or intrathecal (i.t.) routes, but not orally, produced antinociception in the acetylcholine-induced abdominal constriction test (ED50 = 24.8 and 3.6 micrograms, respectively). Likewise, i.c.v. and i.t., but not peripheral (up to 200 mg/kg s.c.), administration increased latency in the 48 degrees C hot-plate test (ED50 = 11.5 micrograms i.c.v. and 0.2 micrograms i.t.). These actions were relatively long-lasting and not blocked by naloxone. Antinociception following i.c.v. administration was abolished in reserpinized mice or by pretreatment with i.t. phentolamine (2 micrograms). These results suggest a moderate antinociceptive action of p-octopamine involving non-opioid, reserpine-sensitive, central pathways.

Evidence that reciprocal hindlimb scratching elicited in mice by intrathecal administration of muscarinic agonists is mediated through M1 type receptors.

Intrathecal (IT) administration of pilocarpine to mice produces a vigorous and dose-related reciprocal hindlimb scratching (RHS) response (ED50 = 0.6 microgram) that is potently blocked by simultaneous IT administration of atropine (ID50 = 0.002 microgram). We now report that RHS is (1) also elicited by the more selective M1 agonist McN-A-343-11 (ED50 = 11.6 micrograms), (2) blocked by the selective M1 antagonist pirenzepine (ID50 = 0.001 microgram), and (3) is not blocked by the selective M2 antagonist AF-DX 116 BS at a dose up to 100 times the ID50 dose of pirenzepine. These results extend our earlier findings and suggest that the RHS elicited in mice by IT injection of muscarinic agonists is mediated through pirenzepine-sensitive (presumably M1) receptors and that RHS may be a convenient in vivo centrally mediated M1 endpoint.

Pilocarpine-induced reciprocal hindlimb scratching in mice.

Intrathecal (IT) administration of pilocarpine (0.25-2.0 micrograms) to mice produced a vigorous and dose-related reciprocal hindlimb scratching response that lasted for 10-15 minutes. Neither the intracerebroventricular administration of pilocarpine at up to 10 times the intrathecal ED90 dose nor the subcutaneous administration of 10 mg/kg pilocarpine caused as robust an effect as IT administration. The reciprocal hindlimb scratching produced by the ED90 dose of pilocarpine (2 micrograms, IT) was antagonized in a dose-related manner by simultaneous IT administration of atropine (ID50 = 0.002 micrograms), methysergide (ID50 = 1.89 micrograms), the substance P antagonist [D-Pro2,D-Trp7,9]-SP (ID50 = 4.94 micrograms), and the putative neurokinin B antagonist [D-Pro2,D-Trp6,8,Nle10]-NK (ID50 = 3.33 micrograms), but not by yohimbine (5 micrograms), phentolamine (2 micrograms), or naloxone (2.5 micrograms). These results suggest that pilocarpine-induced reciprocal hindlimb scratching is mediated spinally, that the effect is produced by an action of pilocarpine on muscarinic receptors in the spinal cord, and that neurokinin, and perhaps 5-HT, mechanisms might also be involved.

Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit.

The morphology of endothelial cells during the induction of atherosclerosis in the descending aortic arch of the hypercholesterol rabbit was studied in situ by scanning electron microscopy (SEM) following silver staining, fixation at physiological pressure, and air-drying of specimens- The earliest deviations from normal endothelial morphology were observed 3 weeks after starting to feed a semi-synthetic diet containing 20% beef fat and 0.2% cholesterol. These were (1) the occurrence of brightly silver stained (argyrophilic) cells, (2) areas of irregularly shaped cells which were often larger and more weakly stained than normal cells and (3) increased incidence of stigmata and stomata associated with the irregular cells. After 6 weeks of hypercholesterolaemia, similar changes were present in the endothelium, but were often also associated with sub-endothelial swelling. These represented the first atherosclerotic lesions. Following 12, 20 and 24 weeks of hypercholesterolaemia, larger raised macroscopic lesions were observed which were always endothelialized. Endothelial morphology and lesion topography suggested that early fatty streaks were composed of numerous focal swellings. In addition to the abnormal endothelial morphology noted at 6 weeks, endothelial cells overlying more advanced lesions became rounded in outline.

Scanning electron microscopy in the evaluation of endothelial integrity of the fatty lesion in atherosclerosis.

The luminal surface of fatty lesions of atherosclerosis was viewed by scanning electron microscopy (SEM). Endothelial cells were outlined by staining intercellular junctions with silver and the aortas were fixed in situ at physiological pressure. When aortas were dehydrated by passage through organic solvents followed by critical point drying from liquid CO2, there was considerable disruption of the luminal surface and it was not possible to correctly interpret the morphological integrity of the endothelium. In contrast, simple air-drying of aortas, without solvent dehydration after fixation, allowed the integrity of the cell layer overlying the lesion to be evaluated. The success of this technique was attributed to the retention of arterial lipids during dehydration of the tissue.