Pertussis toxin-sensitive G(i)-proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery

Abstract

1. We studied the involvement of pertussis toxin (PTX)-sensitive G-proteins in the sensitivity of arterial constriction to intracellular calcium ([Ca(2+)](i)) mobilization. 2. Vasoconstriction was measured in vitro in perfused, de-endothelialized rat tail arteries loaded with the calcium-sensitive dye, fura-2 and treated or not with PTX (30 - 1000 ng ml(-1)). Arteries were stimulated with noradrenaline (NA, 0.1 - 100 microM) or KCl (15 - 120 mM). 3. KCl elicited a smaller vasoconstrictor response (E(max)=94+/-8 mmHg) than NA (E(max)=198+/-9 mmHg) although [Ca(2+)](i) mobilization was similar (E(max)=123+/-8 and 135+/-7 nM for KCl and NA, respectively). PTX (1000 ng ml(-1)) had no effect on [Ca(2+)](i) mobilization but lowered NA- (but not KCl-) induced vasoconstriction (E(max)=118+/-7 mmHg). 4. G(i/o)-proteins were revealed by immunoblotting with anti-G(i alpha) and anti-G(o alpha) antibodies in membranes prepared from de-endothelialized tail arteries. [alpha(32)P]-ADP-ribosylation of G-proteins by PTX (1000 ng ml(-1)) was demonstrated in the intact rat tail artery (pixels in the absence of PTX: 3150, presence: 25053). 5. In conclusion, we suggest that smooth muscle cells possess a PTX-sensitive G(i)-protein-mediated intracellular pathway which amplifies [Ca(2+)](i) sensitivity of contraction in the presence of agonists such as NA.

Figure 1

Typical recordings of changes in perfusion pressure (mmHg) and [Ca²⁺]_i (nM) in rat tail artery segments following KCl (80 mM, 2 min; left) or NA (3 μM, 2 min; right) in the absence (upper) or presence of PTX (300 ng ml⁻¹; lower). Bars represent perfusion with KCl or NA, respectively.

Figure 2

Dose-response curves for increases in perfusion pressure (Δ mmHg, upper), changes in [Ca²⁺]_i (Δ nM, middle) and [Ca²⁺]_i sensitivities of vasoconstriction (lower) following KCl (left) and NA (right) administered in a sequential or a randomized order (n=6–9 per experiment).

Figure 3

Increases in perfusion pressure (Δ mmHg, upper), changes in [Ca²⁺]_i (Δ nM, middle) and [Ca²⁺]_i sensitivities of vasoconstriction (lower) produced by KCl in rat tail artery segments in the absence (dialysis control, n=9) or presence of PTX (30 ng ml⁻¹, n=6; 100 ng ml⁻¹, n=7; 300 ng ml⁻¹, n=7; 1000 ng ml⁻¹, n=6).

Figure 4

Increases in perfusion pressure (Δ mmHg, upper), changes in [Ca²⁺]_i (Δ nM, middle) and [Ca²⁺]_i sensitivities of vasoconstriction (lower) produced by NA in rat tail artery segments in the absence (dialysis control, n=9) or presence of PTX (30 ng ml⁻¹, n=6; 100 ng ml⁻¹, n=7; 300 ng ml⁻¹, n=7, 1000 ng ml⁻¹, n=6). ^*P<0.05 versus dialysis control.

Figure 5

Membrane preparations (10 μg) of rat tail artery labelled with G_iα- (upper) and G_oα (lower)-antibodies. Control was bovine brain purified G-proteins subunits. Lanes: 1, bovine brain purified G-proteins subunits with polyclonal rabbit antibodies directed against G_iα1-2+G_iα3 (upper) or monoclonal mouse antibody directed against G_oα (lower), 2, membrane preparation of the rat tail artery with polyclonal rabbit antibodies directed against G_iα1-2+G_iα3 (upper) or monoclonal mouse antibody directed against G_oα (lower), 3, polyclonal rabbit antibodies directed against G_iα1-2+G_iα3 (upper) or monoclonal mouse antibody directed against G_oα (lower), 4, bovine brain purified G-proteins subunits, 5, membrane preparation of the rat tail artery. The arrows indicate the positions of the molecular markers ovalbumin (45 kDa) and carbonic anhydrase (31 kDa).

Figure 6

Autoradiographs showing ADP-ribosylation of G_i-proteins induced by perfusion (2 h) with PTX (1000 ng ml⁻¹) in rat tail artery. Lanes: 1, rat tail artery perfused with PTX and ADP-ribosylation mixture containing [α³²P]NAD⁺ (30 Ci mmol⁻¹), 2, rat tail artery perfused with ADP-ribosylation mixture containing [α³²P]NAD⁺, 3, rat tail artery perfused with PTX.