Beta1 subunits enable fentanyl‐induced BK channel activation and opioid regulation of cerebral artery diameter

Figures

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  Fig. 1. β1 subunit confers fentanyl sensitivity to calcium‐ and voltage‐gated potassium channels of large conductance (BK). (A) Opioid receptors mu, kappa, and delta (Oprm1, Oprk1, and Oprd1) transcripts are lacking in lysate from non‐transfected Chinese Hamster Ovary (CHO) cells. TaqMan probes used included: Mm01188089_m1 (Oprm1), Mm01230885_m1 (Oprk1), and Mm01180757_m1 (Oprd1). Glyceraldehyde‐3‐phosphate dehydrogenase gene transcript Gapdh (probe Mm99999915_g1) was used as a reference gene. Here and in C, D, error bars indicate standard error of the mean (SEM). (B) Original records showing fentanyl action on currents generated by β1‐lacking (cbv1, top) versus β1‐containing (cbv1 + β1, bottom) calcium−/voltage‐gated potassium channels of large conductance (BK) expressed in CHO cells. Currents were obtained in whole‐cell configuration, Vhold = −80, Vstep = +80 mV, Ca2+ free = 3 μm. Fentanyl was applied from the outside of the cells. (C) Dose–response curve. Here and in C, *P < 0.05 compared to effect on β1‐lacking channels (unpaired t‐test). (D) Dose–response curves normalized to maximal effect of fentanyl across both channel constructs. For β1‐containing channels, data were fitted…

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  Fig. 2. Calcium‐ and voltage‐gated potassium channel of large conductance (BK) β1 subunit participates in fentanyl effect on cerebral artery diameter. (A) Original trace showing changes in middle cerebral artery (MCA) diameter from male wild‐type (top) versus Kcnmb1 −/− mouse (bottom). Here and in C, arteries were pressurized ex vivo using 60 mm Hg intraluminal pressure; dashed lines highlight fentanyl effect on diameter. (B) Averaged concentrations‐response plots from male MCAs showing percent‐change in artery diameter by fentanyl from pre‐fentanyl levels. Data presented as mean ± standard error of the mean (SEM). As the data did not conform to a sigmoidal Boltzmann function, a smooth curve was generated for visualization. n = 6–8 *P < 0.05 by two‐tailed Welch's t‐test. (C) Original trace showing changes in MCA diameter from female wild‐type (top) versus Kcnmb1 −/− mouse (bottom). (D) Averaged concentrations‐response plots from female MCAs showing percent‐change in artery diameter by fentanyl from pre‐fentanyl levels. Data presented as mean ± SEM and fitted using sigmoidal Boltzmann function. n = 6–8 *P < 0.05 by two‐tailed Welch's t‐test.

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  Fig. 3. Opioid receptors mu, kappa, and delta (Oprm1, Oprk1, and Oprd1) transcript presence in C57BL/6J mouse tissue. Spleen and cerebrum were used as positive controls. TaqMan probes used included: Mm01188089_m1 (opioid receptor mu, Oprm1), Mm01230885_m1 (opioid receptor kappa, Oprk1), and Mm01180757_m1 (opioid receptor delta, Oprd1). Glyceraldehyde‐3‐phosphate dehydrogenase gene transcript Gapdh (probe Mm99999915_g1) was used as a reference gene. Gene expression levels were quantified using the 2−ΔCt method [36, 37]. Error bars indicate standard error of the mean (SEM). (A) Data from males. (B) Data from females.

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  Fig. 4. Loss of calcium‐ and voltage‐gated potassium channel of large conductance (BK) β1 subunit unveils fentanyl‐induced constriction in presence of opioid receptors blocker. (A) Original trace showing changes in middle cerebral artery (MCA) diameter from male wild‐type (top) versus Kcnmb1 −/− mouse (bottom). Here and in C, arteries were pressurized ex vivo using 60 mm Hg intraluminal pressure; dashed lines highlight fentanyl effect on diameter. (B) Scattered graphs showing percent‐change in artery diameter from pre‐fentanyl level in male MCAs. Data is shown as mean + standard error of the mean (SEM). n = 5–7. *P < 0.05 by two‐way analysis of variance (ANOVA) for the effect of treatment (10 μm naloxone); #P < 0.01 by two‐way ANOVA for the effect of genotype (loss of BK β1 subunit in global genetic knockout Kcnmb1 −/− ). Here and in D “ns” refers to the lack of statistically significant differences between changes in artery diameter by fentanyl in arteries from Kcnmb1 −/− and wild‐type mice in presence of the BK channel blocker paxilline (1 μm). (C) Original trace showing changes in MCA diameter from female wild‐type (top) versus Kcnmb1 −/− mouse (bottom). (D) Scattered graphs showing…