The Pharmacological Effects of Phenylephrine are Indirect, Mediated by Noradrenaline Release from the Cytoplasm

Abstract

Phenylephrine (PE) is a canonical α₁-adrenoceptor-selective agonist. However, unexpected effects of PE have been observed in preclinical and clinical studies, that cannot be easily explained by its actions on α₁-adrenoceptors. The probability of the involvement of α₂- and β-adrenoceptors in the effect of PE has been raised. In addition, our earlier study observed that PE released noradrenaline (NA) in a [Ca²⁺]_o-independent manner. To elucidate this issue, we have investigated the effects of PE on [³H]NA release and α₁-mediated smooth muscle contractions in the mouse vas deferens (MVD) as ex vivo preparation. The release experiments were designed to assess the effects of PE at the presynaptic terminal, whereas smooth muscle isometric contractions in response to electrical field stimulation were used to measure PE effect postsynaptically. Our results show that PE at concentrations between 0.3 and 30 µM significantly enhanced the resting release of [³H]NA in a [Ca²⁺]_o-independent manner. In addition, prazosin did not affect the release of NA evoked by PE. On the contrary, PE-evoked smooth muscle contractions were inhibited by prazosin administration indicating the α₁-adrenoceptor-mediated effect. When the function of the NA transporter (NAT) was attenuated with nisoxetine, PE failed to release NA and the contractions were reduced by approximately 88%. The remaining part proved to be prazosin-sensitive. The present work supports the substantial indirect effect of PE which relays on the cytoplasmic release of NA, which might explain the reported side effects for PE.

Keywords: Cytoplasmic origin; Indirect action; Noradrenaline release; Noradrenaline transporter; Phenylephrine; α1-Adrenoceptor.

Fig. 1

Effect of prazosin (10 µM) on electrical field stimulation or PE-induced contractions in isolated mouse vas deferens. Contractions induced by PE on mouse vas deferens in the presence of prazosin (A) versus vehicle (B). The organs were allowed to equilibrate under electrical stimulation (trains of 10 Hz with 20 shocks were delivered at 0.1 Hz) for 20–30 min before PE administration. Next, the organ bath was washed out, and the organs were equilibrated once more in the presence of prazosin for 15–20 min. The effect of PE is presented as AUC values (C), which were calculated as the integral of the contraction curve relative to the baseline of the 2 min period for each contraction. The AUC values are presented as the mean ± S.E.M. (C). *: significant difference versus control. Gaussian distribution was assumed following ns. Shapiro–Wilk test (alpha = 0.05). The significance levels were determined by one-way ANOVA followed by Tukey’s post hoc test

Fig. 2

The resting release of [³H]NA induced by PE in a concentration between 0.3 and 30 µM in mouse vas deferens preparation. The release was measured as described in Methods. The preparation was stimulated with supramaximal voltage (10 Hz, 20 shocks) at third fraction. PE was added at different concentrations as indicated. The mean FRR values from fraction 1–8 in each group were compared with the FRR values from fraction 12–19 in that group using paired t-test. PE produced significant increase in NA release in concentrations 0.3, 1, 3, 10, 30 µM (p < 0.05) (A). Note the effect of PE on [³H]NA release is maintained. The concentration of PE is recorded against the effect on resting release (B). The dashed line indicates where the concentration of PE was able to double the extracellular concentrations of NA (approx. 0.8 µM). For calculations of FRR1 and FRR2 see Methods. n = 6 for each group

Fig. 3

Prazosin failed to affect the PE-induced release of [³H]NA (A), the effect of PE is external calcium independent (B). The release was measured as described in Methods. Prazosin was added into the Krebs solution from the 6th fraction and kept in the solution throughout the experiment. PE was added from the 8th fraction. Values are presented as mean ± S.E.M. *: significant difference between groups. Gaussian distribution was assumed following ns. Shapiro–Wilk test (alpha = 0.05). The significance levels were determined by one-way ANOVA followed by Tukey’s post hoc test

Fig. 4

The effect of nisoxetine on electrical field stimulation or PE-induced contractions in isolated mouse vas deferens. Contractions induced by PE on mouse vas deferens in the presence of 10 μM nisoxetine (A), 30 µM nisoxetine (B) or vehicle (C). The organs were allowed to equilibrate under electrical stimulation (trains of 10 Hz with 20 shocks were delivered) for 20–30 min prior to PE administration. Next, the organ bath was washed out, and the organs were equilibrated once more in the presence of nisoxetine for 15–20 min. The effect of PE is presented as AUC values (D), which were calculated as the integral of the contraction curve relative to baseline of the 2 min period for each contraction. The AUC values are presented as the mean ± S.E.M., n = 6 for nisoxetine (10 or 30 µM) or vehicle; n = 14 for control (D). Nisoxetine was present in the Krebs solution from the 6th fraction throughout experiment and inhibited the release of [³H]NA induced by PE (E) measured at 13th and 14th collection periods. For method see legend of Fig. 2E. *: significant difference versus control or between groups as indicated. Gaussian distribution was assumed following ns. Shapiro–Wilk test (alpha = 0.05). The significance levels were determined by one-way ANOVA followed by Tukey’s post hoc test

Fig. 5

Effect of nisoxetine (10 μM) on NA-induced contractions in isolated mouse vas deferens. The effect of noradrenaline is presented as AUC values which were calculated as the integral of the contraction curve relative to the baseline of the 2 min period for each contraction. The AUC values are presented as the mean ± S.E.M., p = 0.7602 (ns). The significance level was determined by two-tailed paired t-test

Fig. 6

The resting release of [³H]NA induced by 10 μM PE in mouse vas deferens preparation in the presence or absence of 10 μM cocaine. The release was measured as described in Methods. The preparation was stimulated with supramaximal voltage (10 Hz, 20 shocks) at third fraction. Cocaine was added into the Krebs solution from the 6th fraction and kept in the solution throughout the experiment. PE was added from the 8th fraction. For calculations of FRR1 and FRR2 see Methods. Statistical analysis was made using two-way ANOVA followed by Tukey’s post-hoc test, the analysis was made between fractions 10–19, n = 6 for each group

Fig. 7

Representative figure of the concentration-dependent contractile effect of PE on smooth muscle under condition in which both vesicular and PE-induced transporter-mediated release of NA are impaired by reserpine pre-treatment and nisoxetine, respectively. The reuptake of NA is also inhibited by nisoxetine contributing to loss of releasable content of vesicles. Reserpinized mouse vas deferens (5 mg/kg i.p. 18 h). Note, the size of contractions induced by train (10 Hz, 20 shocks) at 0.1 Hz reduces by time and PE is still able to contract smooth muscle in a concentration-dependent manner. Take into account that smooth muscles to NA and PE (see Table 1) are supersensitive

Fig. 8

The resting release of [³H]NA induced by 30 µM PE in mouse vas deferens preparation of animals pre-treated with intraperitoneal reserpine (5 mg/kg i.p. 18 h). The preparation was not stimulated. For calculations of FRR1 and FRR2 see the Methods section. n = 5. The release of [.³H]NA in response to PE was taken from Fig. 2A. Statistical analysis was made using two-way ANOVA followed by Tukey’s post-hoc test, n = 5–5. In each fraction except for 10 and 1, NA release between the two depicted groups was significantly different (p < 0.05)

Fig. 9

Mode of action of PE. Role of NAT in vesicular (A) and cytoplasmic (B) release of NA. A NAT controls the temporal and spatial action of released NA by taking back from the extracellular space and NA reused for refilling vesicles. B PE is the substrate of NAT and by means of NAT transported into the cytoplasm together with two Na⁺ and one Cl⁻ ions [45] followed by a counter movement of NA into the extracellular space where it acts on α₁-adrenoreceptors. The effect is large in extracellular concentrations, it does not require axonal activity and Ca²⁺ influx, and hence it is termed non-exocytotic release from noradrenergic boutons without making synaptic contacts [62]. The smooth muscle cells are equipped with highly sensitive α₁-adrenoceptors, this type of non-synaptic receptors are the target of drug treatment [63]. Nisoxetine, a selective NAT inhibitor, inhibits the uptake of NA released in response to action potential (see A) or prevents PE from entering the nerve terminal and the subsequent NA release (B)