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K A Marsh

Publications and source records attributed to K A Marsh.

8 recordsLinked to original sources
PubMed1997

Receptor-stimulated phospholipase C activity in human umbilical artery cultured endothelial cells grown in a low oxygen environment.

Endothelial cells of the human umbilical blood vessels are widely cultured in an oxygen tension (21%) far above that in which they exist in vivo (3%). This study investigates the effect of the long term culture (ca. 1 month) of human umbilical artery endothelial cells in a reduced oxygen environment (3%: HUAEC3) in comparison to cells grown in a 'normoxic' environment (21%: HUAEC21). Despite reports of altered metabolic pathways and reduced membrane integrity in other cell types, the characteristics of HUAEC3 were found to be similar to those of HUAEC21 with respect to morphology, immunocytochemical profile and in vitro growth rates. Cellular glutathione was maintained in these cells although ATP levels in HUAEC3 were found to be significantly lower than those observed in HUAEC21. The phosphoinositide responses of the HUAEC3 to a variety of agonists were also found to be of similar magnitude to those observed in HUAEC21. In addition, the pharmacological characteristics of the phospholipase C-linked histamine H1 and P2y2 (P2U) receptors were not changed by culture of cells in a low oxygen environment.

Adenosine Triphosphate↗
PubMed1996

Desensitization of muscarinic receptor-coupled inositol phospholipid hydrolysis in human detrusor cultured smooth cells.

PURPOSE: The aim of this study was to investigate the desensitization characteristics of muscarinic M3 receptors in primary cultures of human detrusor smooth muscle cells. MATERIALS AND METHODS: Cell cultures were prepared from cold cup pinch biopsies of the human detrusor muscles by explant culture methods. Accumulation of (3)H-inositol phosphates was measured on confluent monolayers as described previously in detail. Desensitization was achieved by preincubating the cells with carbachol or histamine for 5 to 60 minutes. RESULTS: Carbachol induced a concentration-dependent increase in phosphoinositide turnover in naive cells, the response being rapid and evident after only a 30-second exposure to the agonist. Preincubation of the cells with carbachol produced a concentration-dependent decrease in the inositol phosphate response to a second challenge with carbachol. Preexposure to carbachol for only 5 minutes prior to a rechallenge reduced the mean size of response of the second stimulation to 49% of that observed in naive cells. Preexposure of the cells to histamine did not alter the response of the cells to a subsequent challenge with carbachol and vice versa. CONCLUSIONS: The muscarinic receptors retained by human detrusor smooth muscle cells in culture are susceptible to a desensitization of the carbachol-induced increase phosphoinositide turnover observed in these cells. This desensitization is rapid, and the results indicate that it is homologous and does not occur via a postreceptor mechanism but at the level of the receptor itself.

Carbachol↗
PubMed1995

Expression of muscarinic M3-receptors coupled to inositol phospholipid hydrolysis in human detrusor cultured smooth muscle cells.

PURPOSE: To investigate the effect of muscarinic receptor agonists and antagonists on the accumulation of inositol phosphates in cultures of human detrusor smooth muscle cells. MATERIALS AND METHODS: Primary explant culture was used to derive smooth muscle cell lines from small bladder biopsies. The cells were loaded with [3H]-myoinositol, stimulated with muscarinic agonists, and the accumulation of [3H]-inositol phosphates was measured by liquid scintillation counting. RESULTS: Carbachol (EC50 8.3 microM.), methacholine (EC50 7.5 microM.), oxotremorine (EC50 2.5 microM.) and pilocarpine (EC50 8.3 microM.) produced concentration-dependent rises in the accumulation of total [3H]-inositol phosphates. M1 (pirenzepine), M2 (methoctramine) and M3 (4-DAMP and pf-HHSiD) muscarinic receptor antagonists significantly antagonized the response induced by a submaximal concentration of carbachol (100 microM.). The apparent pA2 values were atropine (9.4), 4-DAMP (9.2), pfHHSid (7.4), pirenzepine (6.9) and methoctramine (6.3). CONCLUSIONS: These results indicate that human detrusor smooth muscle cells in culture express M3 muscarinic receptors which are linked to phosphoinositide hydrolysis.

Carbachol↗
PubMed1994

Des-Arg9-bradykinin-induced increases in intracellular calcium ion concentration in single bovine tracheal smooth muscle cells.

1. Dynamic video imaging was used to measure the des-Arg9-bradykinin-induced changes in the intracellular free calcium ion concentration ([Ca2+]i) of single bovine tracheal smooth muscle (BTSM) cells. 2. In the presence of extracellular calcium ions, des-Arg9-bradykinin (1 nM-10 microM) produced a concentration-dependent increase in the [Ca2+]i over basal levels yielding an EC50 value of 316 nM. The percentage of cells responding to each concentration of des-Arg9-bradykinin also increased in a concentration-dependent manner (from 9% to 100%). 3. The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 microM), was without effect on the calcium response of the cells when added 2 min prior to des-Arg9-bradykinin (100 nM). However, the B1 receptor antagonist, des-Arg9Leu8-bradykinin (10 microM), completely abolished the des-Arg9-bradykinin-induced response. 4. Under calcium-free conditions, des-Arg9-bradykinin induced an increase in [Ca2+]i at concentrations of 1 microM and 10 microM. The response to 10 microM des-Arg9-bradykinin was reduced by preincubation of either D-Arg[Hyp3, Thi5,8,D-Phe7]-bradykinin (10 microM) or des-Arg9Leu8-bradykinin (10 microM). 5. We conclude that bradykinin B1 receptors are expressed by cultured BTSM cells and mediate the des-Arg9-bradykinin-induced influx of calcium ions at low agonist concentrations (< 1 microM). At higher concentrations, des-Arg9-bradykinin (1 microM and 10 microM) can stimulate both B1 and B2 receptors to effect intracellular calcium release under calcium-free conditions.

Animals↗
PubMed1993

Isolation and characterization of multiple cell types from a single human colonic carcinoma: tumourigenicity of these cell types in a xenograft system.

The HCA-7 cell line was established from a moderately well differentiated mucinous adenocarcinoma of the colon, which showed histological heterogeneity. This was reflected in the morphological heterogeneity in early passages of the HCA-7 cell line but diminished as the cells were passaged in vitro. Nine subpopulations were isolated from early passage cultures of the HCA-7 line and maintained as cell lines. Each subpopulation demonstrated a unique set of stable biological characteristics in vitro. When established in vivo, there was a wide variation in xenograft generation time. The parent cell line gave rise to six distinct xenograft patterns, two of which had been observed in the primary carcinoma. Individual subpopulations yielded characteristic tumours which were composed of between one and four organizational patterns. Xenografts differed in both the organization of cells and the cell differentiation within the different patterns. Mucous cells were absent from some tumours while abundant in others. The subpopulations isolated from the HCA-7 cell line provide a new and extensive model system for studying the generation and maintenance of phenotypic heterogeneity in colorectal carcinoma.

Adenocarcinoma, Mucinous↗
PubMed1993

Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin. 5. A latency in the response of cells to bradykinin was observed both in calcium-containing and calcium-free conditions.6. We conclude that bradykinin B2 receptors are expressed by BTSM cells and are involved in the bradykinin-induced increase in [Ca2+]i. It appears that the increase in [Ca2+], can be mediated via a graded influx of calcium ions from the extracellular space or an 'all-or-nothing' release from intracellular stores.

Adamantane↗
PubMed1992

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.

Animals↗