Further investigations into adenosine A1 receptor-mediated contraction in rat colonic muscularis mucosae and its augmentation by certain alkylxanthine antagonists.

1. The alkylxanthine antagonists, 8-phenyltheophylline (8-PT), 8-p-sulphophenyltheophylline (8-SPT) and 1,3,7-trimethylxanthine (caffeine) produced rightward displacements of contractile concentration-effect curves to 5'-N-ethylcarboxamidoadenosine (NECA) in rat isolated colonic muscularis mucosae (RCMM) with concentration-ratios consistent with adenosine receptor blockade. The non-xanthine antagonist, 9 fluro-2-(2-furyl)-5,6-dihydro [1,2,4] triazo to [1,5-c]-quinazin-imine (CGS15943A) also antagonized contractions to NECA with an affinity (pKB8.1-8.5) consistent with adenosine A1 receptor blockade. 2. In addition to producing rightward shifts of the concentration-response curves, the maximum contractions to 5'-N-ethylcarboxamidoadenosine (NECA) were also markedly increased in the presence of 8-PT (by 83 +/- 16% at 1 microM), 8-SPT (by 37 +/- 7% at 10 microM) and caffeine (by 45 +/- 5% at 100 microM) but were unaffected by CGS15943A (at 0.01 and 0.03 microM). 3. As with NECA, the maximum contractions to the adenosine A1 receptor agonists R-phenylisopropyladenosine (R-PIA) and N-[(1S, trans)-2-hydroxyclopentyl] adenosine (GR79236) were both antagonized and augmented by 8-PT. In addition, the contractions to NECA in the presence of 8-PT (1 microM) were inhibited by nanomolar concentrations of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (1 microM) produced a marked increase in the NECA maximum without producing a rightward shift in the NECA curve, whereas a higher concentration (10 microM) virtually abolished responses. The PDE type III inhibitor,milrinone (1 microM), the type IV inhibitor, rolipram (10 microM), and the type V PDE inhibitor, zaprinast(3 microM), were all without effect on NECA responses in RCMM.5. Partial inhibitions of contractions to NECA were produced by indomethacin (at 3 or 10 micro M) or piroxicam (at 3 microM). Responses to GR79236 were also partially inhibited by indomethacin. In the presence of indomethacin, 8-PT was still able to enhance markedly the maximum contractions obtained to NECA in RCMM.6. The present study has shown that certain alkylxanthine antagonists (but not the non-xanthineCGS15943A) produced a marked augmentation of adenosine Al receptor-mediated contractions inRCMM. The mechanism of this augmentation is, as yet, not known but is unlikely to result from inhibition of PDE. This study has also shown that adenosine Al receptor-induced contractions inRCMM are mediated, in part, via products of the cyclo-oxygenase pathway.

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae.

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol. 5. The contractile responses to NECA in RCMM were not affected by atropine (1 microM), tetrodotoxin(0.3 microM) or the P2 antagonist, suramin (100 microM).6. The present study confirms that contractions to adenosine agonists in the RCMM are mediated via adenosine Al receptors.

Electromyographic and mechanical twitch responses following suxamethonium administration.

Electromyographic and mechanical evoked twitch responses at the hand were observed simultaneously following suxamethonium. The intensity of neuromuscular blockade was assessed by comparison with their control heights of the responses obtained from both the force of thumb adduction and from the compound electromyogram (EMG). Surface electrodes placed dorsally over the lateral hand provided two EMG signals, one of which was assessed in terms of its amplitude, the other as the rectified and integrated energy envelope. While the mechanical and electrical events were roughly in agreement there was better conformity between the two EMG techniques in the assessment of paralysis.

Cytogenetics of malignant epithelial cells and lymphoblastoid cell lines from nasopharyngeal carcinoma.

The malignant epithelial cells of nasopharyngeal carcinoma (NPC) and cells of lines derived form the lymphoid cells which infiltrate this tumour have been investigated cytogenetically. Chromosome spreads of lymphoblastoid cells of lines established from 7 different NPC biopsy specimens were examined after banding staining. Banding was also applied to the epithelial tumour cells of 5 further biopsy specimens freed from non-malignant infiltrating cells by passage through nude mice; epithelial cell spreads were obtained by in vivo splindle arrest. Five of the lymphoblastoid lines were found to be diploid, and 2 tetraploid; the karyotypes were essentially normal. The squamous epithelial nature of the cells in the nude-mouse-grown NPC tumours was established by light and electronmicroscopy, and 3 tumours were found to be near-triploid, and 2 near-diploid. The cells of the near-triploid tumours contained grossly abnormal chromosomes but those of the near-diploid tumours showed only relatively minor changes. Although abnormalities were observed which were specific for cells from each individual tumour, no discernible change was common to cells from all the tumours.

Cytogenetic observations on the malignant epithelial cells and infiltrating lymphocytes of nasopharyngeal carcinoma.

Because of the presence of a No 14 chromosome marker abnormality in EB virus-carrying cells from African Burkitt's lymphoma (BL) but not from other origins, and the association of EB virus with nasopharyngeal carcinoma (NPC) as well as BL, both the malignant epithelial cells and the non-malignant lymphoid cells of NPC have been investigated cytogenetically. Chromosome spreads from seven NPC-derived lymphoblastoid lines were examined after banding; five lines were diploid and two were tetraploid. No consistent no. 14 chromosome abnormalities were found. Apart from a secondary constriction near the centromere of both no. 1 chromosomes in all cells of one diploid line, no consistent significant abnormalities were seen. Five NPC tumours, freed of infiltrating lymphoid cells by passage through nude mice, were similarly examined after spindle arrest in vivo. Two tumours were near-diploid, and three were hypo-tetraploid. Near-diploid cells had only minor chromosomal changes, but the hypotetraploid spreads from all tumours showed gross changes of uncertain origin, including frequent, major translocations. No. 14 chromosome marker changes were not seen, and there was no other consistent pattern of abnormality in tumour cells.

Observations on the type of infection by Epstein-Barr virus in peripheral lymphoid cells of patients with infectious mononucleosis.

Transformation to continuous cell lines has been studied in cultures of peripheral leuckocytes from infectious mononucleosis (IM) patients and in co-cultures of IM leukocytes and foetal cord blood leukocytes of opposite sex. The transformed cells in the co-cultures were of mixed origin with foetal cells usually predominating. Neutralizing antisera to EB virus markedly reduced or abolished the incidence of transformation in IM leukocyte cultures. This effect was not due to cytotoxicity and followed the pattern seen with cultures where transformation was known to depend on the inter-cellular transfer of infectious EB virus. The findings suggest that EB virus is harboured in peripheral lymphocytes of IM patients as a non-productive unexpressed infection which is activated to produce virus in vitro, the particles released then infecting neighbouring cells to give transformed lines. The differences between this mechanism and the one whereby lines arise in culture from malignant cells of Burkitt's lymphoma are considered, and their significance is discussed.

Cytogenetic studies on human lymphoblastoid cell lines from Burkitt's lymphomas and other sources.

The occurrence and incidence of a characteristic banding abnormality in a No. 14 chromosome has been studied in Burkitt-lymphoma-derived and certain other EB virus-associated lymphoblastoid cell lines. The abnormality was readily detected in 7 out of 7 Burkitt lines, and when present could be seen in 100% of cells with recognizable No. 14 chromosomes. In contrast, the abnormality was not observed in 775 cells from 31 infectious mononucleosis-derived lines nor in 450 cells from 18 lines obtained from cord blood lymphocytes experimentally transformed by EB virus in vitro. The significance of the abnormality as an indication of cells transformed by the virus in vivo is discussed, and the importance of this considered in relation to a possible oncogenic role for EB virus in man.

An unusual karyotypic observation on cultured cells from an owl monkey (Aotus trivirgatus).

A cell line, established from a pathological lymph node of an owl monkey, Aotus trivirgatus s.sp. trivirgatus, was found to have two cell populations distinguished from one another by chromosome number. The normal karyotype was present in the population of cells with 54 chromosomes; the other cell population, with 55 chromosomes, possessed a normal karyotype but with one additional abnormal chromosome. The significance of this extra chromosome is discussed.