Uptake of Adenosine by Cultured Cerebral Vascular Smooth Muscle Cells

Beck, David; Vinters, Harry V.; Moore, Steven A.; Hart, Michael N.; Cancilla, Pasquale A.

doi:10.1111/j.1471-4159.1983.tb09037.x

Cited by 14 publications

(6 citation statements)

References 16 publications

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“…The NBMPR-sensitive transport of adenosine by smooth muscle cells measured in this study exhibited a low affinity (Km = 190 ìÒ), reportedly a characteristic of peripheral tissues (Mun et al 1998;Clanachan & Parkinson, 1990;Baldwin et al 1999), and compares with Km estimates (up to 500 ìÒ) from other studies of overall adenosine uptake performed in cultured cells or perfused tissues (Olsson et al 1972;Pearson et al 1978;Bakhle & Chelliah, 1983;Catravas , 1984). The apparent Km value for adenosine transport in human smooth muscle cells was similar to values reported for system es in human umbilical vein endothelial cells (HUVEC; Km = 80 ìÒ; Sobrevia et al 1994) and T84 intestinal epithelial cells (Km = 114 ìÒ;Mun et al 1998), but was •19-fold higher than values characterized in cerebral vessels (Km = 10 ìÒ; Beck et al 1983), the smooth muscle cell line DDT1 MF-2 (Km = 9.5 ìÒ, Parkinson et al 1996), or in bovine adrenal medulla endothelial cells (BAMEC Km = 10 ìÒ; Sen et al 1996). This suggests that intrinsic properties of adenosine transporters in human umbilical artery smooth muscle may be different from other cell types.…”

Section: Discussionsupporting

confidence: 79%

“…At least two equilibrative nucleoside transporter isoforms have been identified: system es (inhibited by nanomolar nitrobenzilthioinosine, NBMPR) and system ei (inhibited by micromolar NBMPR) (for review, see Baldwin et al 1999). It has been shown that adenosine transport is mediated by a high affinity system (Km 1-10 ìÒ) in pig aorta (Pearson et al 1978) and rat brain (Beck et al 1983) smooth muscle cells and in the Syrian hamster vas deferens smooth muscle cell line DDT1 MF-2 (Foga et al 1996;Borgland & Parkinson, 1998), but there are no reports on adenosine transport in human vascular smooth muscle. Diabetes mellitus is a disease characterized by high plasma levels of ª_glucose, where modulation of vascular tone is altered and a lowered sensitivity of vascular smooth muscle cells to endothelium-derived NO has been suggested (for reviews, see Poston & Taylor, 1995;Baron, 1999).…”

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confidence: 98%

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Nitric Oxide, cGMP and cAMP Modulate Nitrobenzylthioinosine‐Sensitive Adenosine Transport in Human Umbilical Artery Smooth Muscle Cells from Subjects with Gestational Diabetes

Aguayo

Sobrevía

2000

Experimental Physiology

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Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from non-diabetic and diabetic pregnant subjects. Transport of adenosine was mediated by a Na+-independent transport system inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) in both cell types. Diabetes increased adenosine transport, an effect that was associated with a higher maximal velocity (Vmax) for NBMPR-sensitive (es) saturable nucleoside transport (18 +/- 2 vs. 61 +/- 3 pmol (microgram protein)-1 min-1, P < 0.05) and the maximal number of binding sites (Bmax) for specific [3H]NBMPR binding (74 +/- 4 vs. 156 +/- 10 pmol (microgram protein)-1, P < 0.05), with no significant changes in the Michaelis-Menten (Km) and dissociation (Kd) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 microM NG-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (with 1 microM cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 microM, SQ-22536) in non-diabetic cells. Diabetes-induced adenosine transport was blocked by L-NAME and associated with an increase in L-[3H]citrulline formation from L-[3H]arginine and intracellular cGMP, but with a decrease in intracellular cAMP compared with non-diabetic cells. Expression of inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP (dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine transport in non-diabetic cells. dbcGMP or the NO donor S-nitrosoacetylpenicillamine (SNAP, 100 microM) did not alter the diabetes-elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 microM), directly or after incubation of cells with dbcAMP, inhibited adenosine transport in both cell types. Our findings provide the first evidence that adenosine transport in human umbilical artery smooth muscle cells is mediated by the NBMPR-sensitive transport system es, and that its activity is upregulated by gestational diabetes.

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Section: Discussionsupporting

confidence: 79%

mentioning

confidence: 98%

Nitric Oxide, cGMP and cAMP Modulate Nitrobenzylthioinosine‐Sensitive Adenosine Transport in Human Umbilical Artery Smooth Muscle Cells from Subjects with Gestational Diabetes

Aguayo

Sobrevía

2000

Experimental Physiology

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“…2-Chloroadenosine is structurally similar to adenosine and has been reported to both block transport and be transported in other cell systems (4,44). Smooth muscle cells from the same preparation were exposed to either 10 M adenosine (spiked with [ 3 H]adenosine) or 10 M 2-CAD (spiked with 2-chloro-[ 3 H]adenosine), and uptake was measured in the presence and absence of NBTI after 10 min incubation.…”

Section: Effect Of Adenosine Transport Inhibitors On Adenosine-mediatmentioning

confidence: 99%

“…The relatively low uptake of adenosine into coronary smooth muscle compared with other cell types (3) also reinforced the conclusion that vascular smooth muscle played a minor role in adenosine regulation. One brief report (4), however, showed that cultured cerebral vascular smooth muscle accumulated adenosine with a high affinity, but data were not presented concerning sensitivity to selective blockade. It is not established whether such a transporter is expressed in vascular smooth muscle in native arteries or in acutely isolated cells not exposed to growth factors during cell culture.…”

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confidence: 99%

Selective transport of adenosine into porcine coronary smooth muscle

Rubin

Johnson²,

Dodam³

et al. 2000

American Journal of Physiology-Heart and Circulatory Physiology

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Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

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“…The action of adenosine with the brain microvasculature is undoubtedly complex and its interaction with specific microvascular elements is unclear. It has been shown that adenosine is taken up by isolated micro vessels, 23 cerebral endothelium 24 and cerebral smooth muscle 25 via a carrier-mediated system. Also, the affinity of uptake into isolated cerebral endothelium (Km = 5.0 /xM) is greater than that into isolated cerebral smooth muscle cells (Km = 10.0 jiM).…”

Section: Resultsmentioning

confidence: 99%

Demonstration of adenosine receptors on mouse cerebral smooth muscle membranes.

Beck¹,

Vinters²,

Moore³

et al. 1984

Stroke

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Adenosine receptors have been identified on brain cortical membranes and microvascular preparations. However, they have not been demonstrated on specific microvascular elements in isolation. 2-3H-chloroadenosine was used as a ligand to investigate the presence of adenosine receptors on isolated mouse cerebral smooth muscle membranes. The binding studies reveal the presence of a high affinity binding site with a Kd value of 33.3 nM and a maximal binding capacity (Bmax) of 283 fmol/mg protein. These findings demonstrate that there is an adenosine receptor on cerebral smooth muscle membranes.

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Uptake of Adenosine by Cultured Cerebral Vascular Smooth Muscle Cells

Cited by 14 publications

References 16 publications

Nitric Oxide, cGMP and cAMP Modulate Nitrobenzylthioinosine‐Sensitive Adenosine Transport in Human Umbilical Artery Smooth Muscle Cells from Subjects with Gestational Diabetes

Nitric Oxide, cGMP and cAMP Modulate Nitrobenzylthioinosine‐Sensitive Adenosine Transport in Human Umbilical Artery Smooth Muscle Cells from Subjects with Gestational Diabetes

Selective transport of adenosine into porcine coronary smooth muscle

Demonstration of adenosine receptors on mouse cerebral smooth muscle membranes.

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