Figure 1. Extracellular nucleotides promote vasoconstriction, Ca_V1.2 activity and Ser¹⁹²⁸ phosphorylation in response to 20 mM D-glucose in murine arterial myocytes.

(A) Representative diameter recordings and summary arterial tone data from pressurized (60 mmHg) wt mouse cerebral arteries before and after application of 20 mM D-glucose in the absence (n = 6 arteries from six mice) and presence (n = 6 arteries from six mice) of apyrase (apy; 0.32 U/ml; *p<0.05, Wilcoxon matched pairs test; Figure 1—source data 1). (B) Characteristic I_Ba recordings from the same cell and summary I_Ba data from wt mouse cerebral arterial myocytes evoked by step depolarizations from −70 to +10 mV before and after application of 20 mM D-glucose in the absence (n = 11 cells from five mice) and presence of apyrase (n = 9 cells from five mice) (*p<0.05, paired t test; Figure 1—source data 2). (C) Representative immunoblot detection of phosphorylated Ser¹⁹²⁸ (pSer¹⁹²⁸) and total Ca_V1.2 from wt mouse cerebral and mesenteric arteries after 10 min incubation with 10 mM or 20 mM D-glucose in the absence and presence of apyrase (n = 10 arterial lysates per condition), and quantification of pSer¹⁹²⁸ (AU = arbitrary units) (*p<0.05, Kruskal-Wallis with Dunn’s multiple comparisons; Figure 1—source data 3).

Figure 1—figure supplement 1. K⁺-induced arterial constriction in the absence and presence of apyrase, no changes in arterial tone, I_Ba and pSer¹⁹²⁸ in response to 20 mM mannitol, and full-length blots corresponding to Figure 1C.

(A) Bar plot of % constriction in response to high K⁺ (60 mM) from wt mouse cerebral arteries exposed to 10 mM D-glucose in the absence and presence of apyrase (apy; 0.32 U/ml; n = 6 arteries from six mice per condition; Figure 1—figure supplement 1—source data 1). Response to high K⁺ (60 mM) was obtained in pressurized arteries at 20 mmHg. (B) Representative diameter recording and summary arterial tone data from pressurized (60 mmHg) wt mouse cerebral arteries before and after application of 20 mM mannitol (n = 6 arteries from six mice; Figure 1—figure supplement 1—source data 2). (C) Exemplary I_Ba recording from the same cell and summary I_Ba data from wt mouse cerebral arterial myocytes evoked by step depolarizations from −70 to +10 mV before and after application of 20 mM mannitol (n = 7 cells from three mice; Figure 1—figure supplement 1—source data 3). (D) Complete scan of representative phosphorylated Ser¹⁹²⁸ (pSer¹⁹²⁸) and total Ca_V1.2 blots for mouse arteries incubated with 10 mM D-glucose, 20 mM D-glucose and 20 mM D-glucose +apyrase. Red boxes indicate the crop region displayed in the main Figure 1C. (E) Representative immunoblot detection of phosphorylated Ser¹⁹²⁸ (pSer¹⁹²⁸) and α-tubulin (loading control) from wt mouse arteries after 10 min incubation in either 10 mM D-glucose or 20 mM mannitol (n = 5 arterial lysates from five mice per condition) and quantification of pSer¹⁹²⁸ (AU = arbitrary units) (*p=0.3835, unpaired t-test; Figure 1—figure supplement 1—source data 4).

Figure 1—figure supplement 2. Enhanced I_Ba in response to elevated glucose is prevented by continuous bath perfusion.

(A) Representative I_Ba recordings from the same cell and (B) summary I_Ba data from wt mouse cerebral arterial myocytes induced by step depolarizations from −70 to +10 mV during exposure to 10 mM D-glucose and in response to 20 mM D-glucose with constant bath perfusion (flow) and after stopping perfusion (e.g. static) (n = 9 cells from three mice; *p<0.05, Friedman Test with Dunn’s multiple comparisons; Figure 1—figure supplement 2—source data 1). For these experiments, cells were patched in a bath solution containing 10 mM D-glucose at a perfusion rate of 2.1 mL/min. After establishment of a stable gigaseal for at least 5 min, I_Ba were recorded in the presence of 10 mM D-glucose under continuous flow. Cells were then perfused with a bath solution containing 20 mM D-glucose under continuous flow for at least 5 min before recording of I_Ba again. Subsequently, the bath perfusion was stopped, and cells were bathed in the 20 mM D-glucose solution under static bath condition for five more minutes before recording I_Ba one more time. If the three experimental conditions could not be performed in the same cells, the results were discarded.