![Figure 9. Lack of constitutive Ca2+ channel activity in vascular myocytes. (A) In single portal vein myocytes, depolarization (−70 to +10 mV; vi) activated a voltage-dependent Ca2+ current (ICa; v) to evoke a rise in [Ca2+] in the subplasma membrane space (ii) and bulk cytoplasm (iii). 1 µM nimodipine (iv) applied from a puffer pipette (A, i, left panel, Nimodipine Pipette) did not alter the resting subplasma membrane (ii) or bulk average (iii) [Ca2+] but blocked ICa (v) and the accompanying [Ca2+] rise (ii and iii). The break in the record is ∼3 min after which ICa and [Ca2+] partially recovered. A bright field image of the cell is shown (A, i, left panel); see also whole cell electrode (right side). (B) In single portal vein myocytes, hyperpolarization (−130 mV; vi) from the resting potential (−70 mV; vi) evoked an increase in [Ca2+]PM (ii) and [Ca2+]c (iii). 1 µM nimodipine (iv), an inhibitor of voltage-dependent Ca2+ channels, applied by pressure ejection from a puffer pipette (B, i, left panel) did not reduce the hyperpolarization-evoked [Ca2+]c or [Ca2+]PM rise. Changes in the fluorescence ratio with time (A, ii and iii, and B, ii and iii) are derived from the regions encompassing that part of the cell in TIRF (A, i, and B, i, right-hand panels). (C) PKCα expression in portal vein. The immunoblot shows increasing loads of total protein (10, 15, and 20 µg in lanes 1–3) from guinea pig portal vein whole homogenate, which was probed with mouse anti-PKCα monoclonal IgG1. The position of molecular weight markers run alongside is indicated. The result is typical of those obtained from two other independent experiments. (D) CaV1.2 expression in guinea pig portal vein. Immunoblot showing increasing loads of total protein (10, 20, 30, and 40 µg in lanes 1–4) from guinea pig portal vein whole homogenate, which was probed with rabbit anti-CaV1.2 polyclonal antibody. Lane 5 shows 10 µg cardiac ventricular homogenate used here as a positive control for CaV1.2. The position of molecular weight markers run alongside is indicated. Results shown are typical of two other experiments.](https://cdn.rupress.org/rup/content_public/journal/jgp/133/4/10.1085_jgp.200810189/5/jgp_200810189_rgb_fig9.jpeg?Expires=1773551391&Signature=Xux1-~0n~du0f6bXKDEIkjfqa~BqywPx7L1qrgT2~Clx5m~y3xDLPSpxXbT2vE-qztKofUCHhAN1x8eLOnQpY9J62fZJX3GQh~FPg5aX499aBdYY4PjJ2pQfexVp6lnbcDD9q-gpym8ieIlPATfFzTQE1AHnpmrYkLowIi6I9gCmEUERTwyVod4nRNdncpKbJevpztApsJmynASaysgLpFmuuEksv-b2swWn-5H6taycbyQ7-47YOpmqxYS-BxLLPOlyoooRihY4mOQKnMSS9gfMiNJaZcP1eHm8Prcj43JjkAP8ahvoWniMwlmWTwsgkXpRGUdZRAtl1USa8wDypg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Lack of constitutive Ca2+ channel activity in vascular myocytes. (A) In single portal vein myocytes, depolarization (−70 to +10 mV; vi) activated a voltage-dependent Ca2+ current (ICa; v) to evoke a rise in [Ca2+] in the subplasma membrane space (ii) and bulk cytoplasm (iii). 1 µM nimodipine (iv) applied from a puffer pipette (A, i, left panel, Nimodipine Pipette) did not alter the resting subplasma membrane (ii) or bulk average (iii) [Ca2+] but blocked ICa (v) and the accompanying [Ca2+] rise (ii and iii). The break in the record is ∼3 min after which ICa and [Ca2+] partially recovered. A bright field image of the cell is shown (A, i, left panel); see also whole cell electrode (right side). (B) In single portal vein myocytes, hyperpolarization (−130 mV; vi) from the resting potential (−70 mV; vi) evoked an increase in [Ca2+]PM (ii) and [Ca2+]c (iii). 1 µM nimodipine (iv), an inhibitor of voltage-dependent Ca2+ channels, applied by pressure ejection from a puffer pipette (B, i, left panel) did not reduce the hyperpolarization-evoked [Ca2+]c or [Ca2+]PM rise. Changes in the fluorescence ratio with time (A, ii and iii, and B, ii and iii) are derived from the regions encompassing that part of the cell in TIRF (A, i, and B, i, right-hand panels). (C) PKCα expression in portal vein. The immunoblot shows increasing loads of total protein (10, 15, and 20 µg in lanes 1–3) from guinea pig portal vein whole homogenate, which was probed with mouse anti-PKCα monoclonal IgG1. The position of molecular weight markers run alongside is indicated. The result is typical of those obtained from two other independent experiments. (D) CaV1.2 expression in guinea pig portal vein. Immunoblot showing increasing loads of total protein (10, 20, 30, and 40 µg in lanes 1–4) from guinea pig portal vein whole homogenate, which was probed with rabbit anti-CaV1.2 polyclonal antibody. Lane 5 shows 10 µg cardiac ventricular homogenate used here as a positive control for CaV1.2. The position of molecular weight markers run alongside is indicated. Results shown are typical of two other experiments.
