Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways

Abstract

Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca(2+) from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca(2+) signaling pathway activated by 10-100 pM AVP. This pathway is distinguished from the PLC pathway by its dependence on protein kinase C (PKC) and L-type voltage-sensitive Ca(2+) channels (VSCC). In the present study, we used isolated, pressurized rat mesenteric arteries to examine the contributions of these different Ca(2+) signaling mechanisms to AVP-induced vasoconstriction. AVP (10(-14)-10(-6) M) induced a concentration-dependent constriction of arteries that was reversible with a V(1a) vasopressin receptor antagonist. Half-maximal vasoconstriction at 30 pM AVP was prevented by blockade of VSCC with verapamil (10 microM) or by PKC inhibition with calphostin-C (250 nM) or Ro-31-8220 (1 microM). In contrast, acute vasoconstriction induced by 10 nM AVP (maximal) was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min, the remaining vasoconstriction induced by 10 nM AVP was partially dependent on PKC activation and almost fully dependent on VSCC. These results suggest that different Ca(2+) signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca(2+) signaling pathway that is dependent on PKC activation and can be inhibited by Ca(2+) channel blockers.

Fig. 1

A: representative real-time tracing of the outer diameter of a mesenteric artery exposed to cumulative concentrations of [Arg⁸]vasopressin (AVP). Time is plotted on the x-axis (minutes), while vessel outer diameter (μm) is represented on the y-axis. B: cumulative concentration-dependent vasoconstrictor effects of AVP. Results from 6 experiments are represented as means ± SE. *Significant vasoconstrictor effect vs. baseline (P < 0.05).

Fig. 2

Both low and high concentrations of AVP exert vasoconstrictor effects via V_1a vasopressin receptors. Treatments are indicated by shaded or hatched boxes above the trace. Blank areas indicate control time periods or periods immediately following the washout of drugs. A: after a stable 1-h recording of baseline diameter, constrictor responses to 30 pM and 10 nM AVP, as well as 60 mM KCl, were completely reversed by superfusion washout. Inset: images of a pressurized artery; a, b, and c illustrate vessel diameters during baseline and stable constrictor responses to 30 pM and 10 nM AVP, respectively. B: the vasoconstrictor effects of 30 pM and 10 nM AVP were completely reversed by addition of a V_1a vasopressin receptor antagonist. Summarized results are shown in Fig. 6.

Fig. 3

Blockade of voltage-sensitive L-type Ca²⁺ channels significantly attenuates 30 pM AVP-induced vasoconstriction but not 10 nM AVP-induced constriction. Arteries were preincubated with 10 μM verapamil for 5 min and then throughout the experiment. Summarized results are shown in Fig 6.

Fig. 4

Inhibition of protein kinase C (PKC) abolishes 30 pM AVP-induced vasoconstriction, but responses to 10 nM AVP are not inhibited. Pressurized arteries were preincubated with the PKC inhibitor Ro-31-8220 (1 μM) for 30 min and then throughout the experiment. Application of 30 pM AVP did not generate vasoconstriction, whereas 10 nM AVP induced significant vasoconstriction. Summarized results are shown in Fig 6.

Fig. 5

PKC activation leads to vasoconstriction via activation of L-type Ca²⁺ channels. Addition of the PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA, 100 nM), to the vessel bath induced significant vasoconstriction (*P < 0.05). Once constriction had reached a plateau with PMA, verapamil (10 μM) was added to the arterial bath and significantly reversed PMA-induced vasoconstriction (P < 0.05). Results are means ± SE for 4 experiments.

Fig. 6

Concentration-dependent switch in signal transduction for AVP-induced vasoconstriction. Summarized results are shown, normalized to a percentage of maximal outer diameter. 30 pM AVP induced significant vasoconstriction, which was completely reversed with a V_1a vasopressin receptor antagonist, and significantly inhibited by the voltage-sensitive L-type Ca²⁺ channel blocker, verapamil (Verap, 10 μM), as well as the PKC inhibitors, calphostin-C (Cal-C, 250 nM) and Ro-31-8220 (Ro, 1 μM) (*P < 0.05). 10 nM AVP induced significant vasoconstriction, which was completely reversed by the V_1a vasopressin receptor antagonist (*P < 0.05), but was not affected by verapamil or PKC inhibitors.

Fig. 7

Acute vasoconstriction induced by 10 nM AVP was similar in the presence or absence of verapamil (10 μM) or calphostin-C (250 nM). *All arteries significantly relaxed after 30 min of 10 nM AVP exposure (P < 0.05). Arteries preincubated with verapamil or calphostin-C relaxed significantly more than control arteries at 30 min (‡P < 0.001 and P = 0.011), respectively. †Acute addition of verapamil 30 min after 10 nM AVP induced significant relaxation in the presence or absence of calphostin-C (P < 0.05). There was also slightly, but significantly, more relaxation in verapamil-treated arteries preexposed to calphostin-C vs. control arteries (#P = 0.014).

Fig. 8

Hypothetical model for AVP concentration-dependent transitions between Ca²⁺ entry and Ca²⁺ release. Picomolar AVP concentrations induce voltage-sensitive Ca²⁺ entry via PKC activation and inhibition of voltage-activated K⁺ channels (K_v) (light shaded box). Nanomolar concentrations of AVP acutely activate the well-established AVP signaling pathway, which is dependent on phospholipase C (PLC) activation and inositol 1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ release (dark shaded box). Prolonged nanomolar AVP stimulation (30 min) activates nonselective cation channels (NSCC), via diacylglycreol (DAG), which leads to membrane depolarization (ΔE_m) and voltage-sensitive Ca²⁺ entry. Inhibition of PKC would block downstream signaling pathways (dashed lines). V_1a, V_1a vasopressin receptor; PLD, phospholipase D; VSCC, voltage-sensitive L-type Ca²⁺ channels; G_q, G protein alpha q subunit.