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12.4.2 Purinoceptor-Mediated Ca
2+
Rise in Isolated Islet
Endothelial Cells and
β
-Cells
It was possible to compare the Ca
2+
responses of islet endothelial cells and
β
-cells
after dissociation of
-cell rich ob/ob mouse islets [34] into single cells and small
aggregates [37]. Suspensions of islet cells were allowed to attach to coverslips dur-
ing culture in medium supplemented with endothelial cell growth factor (ECGF), to
maintain the phenotype of the endothelial cells [59]. [Ca
2+
]
i
was measured during
perifusion with ratiometric fura-2 technique [37, 59]. Endothelial cells, prelimi-
nary identified by addition of Dynabeads coated with the
Bandeira simplicifolia 1
lectin [60], responded in a similar way as those stained with CD31 antibodies after
measurements of [Ca
2+
]
i
.
Endothelial cells differed from
β
-cells by lacking spontaneous fluctuations of
[Ca
2+
]
i
The ability of different test substances to generate [Ca
2+
]
i
rises in the
presence of 5 mM glucose is shown in Table 12.2 and Fig. 12.4. Both endothe-
lial cells and
β
M ATP with rise of [Ca
2+
]
i
.Inthe
β
-cells responded to 10
μ
M UTP was equally effective as ATP in raising [Ca
2+
]
i
but equimolar ADP, UTP, adenosine or acetylcholine lacked effect. Studying the
responses of the
endothelial cells 10
μ
M ADP and acetylcholine initi-
ated rise of [Ca
2+
]
i
, but UTP, UDP, adenosine and acetylcholine were without
effect.
Subsequent fura-2 measurements confirmed that UTP was equally effective as
ATP in raising [Ca
2+
]
i
in islet endothelial cells (Figs. 12.5 and 12.6). The obser-
vation that ATP lacks effects in the presence of UTP (Fig. 12.5a) and that both
nucleotides elicit [Ca
2+
]
i
rises with similar kinetics, suppressed with 100
β
-cells it was found that 10
μ
M
suramin (Fig. 12.5b), but not with methoxyverapamil (Fig. 12.6), suggest that most
of their actions are mediated by the same type of purinoceptor, probably P2Y
2
.
Additional binding to P2Y
4
and P2Y
6
receptors seems unlikely, since these recep-
tors are relatively resistant to suramin inhibition [19]. Moreover, there was no
increase of [Ca
2+
]
i
when the endothelial cells were exposed to UDP, the princi-
pal activator of P2Y
6
receptors. In many types of endothelial cells the ATP- induced
increase of [Ca
2+
]
i
is mediated by activation of P2Y
1
receptors [24, 63]. However,
there was no [Ca
2+
]
i
response to ADP, indicating that mouse islet endothelium lacks
μ
Table 12.2
Rise of
cytoplasmic Ca
2+
in mouse
islet endothelial cells and
β
-cells in the presence of
5mMD-glucose
Cell
Endothelial cell
Beta-cell
ATP ; 1 0
μ
M
+
+
ADP; 10
μ
M
0
+
UTP; 10
μ
M
+
0
UDP; 10
μ
M
0
0
Adenosine; 10
μ
M
0
0
Acetylcholine; 10
μ
M0
+
Results from reference [37]
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