L-tryptophan ethyl ester dilates small mesenteric arteries by inhibition of voltage-operated calcium channels in smooth muscle

Abstract

Background and purpose: L-tryptophan (L-W) is a precursor of the vasoconstrictor, 5-HT. However, acute administration of L-W ethyl ester (L-Wee) lowered blood pressure. The mechanism of action is unknown. This study compares the vascular effects of L-W and L-Wee in intact animals, isolated aortic rings, small mesenteric arteries (MA) and explores possible mechanisms by studies in vascular smooth muscle cells (VSMC) of MA.

Experimental approach: Effects of L-W or L-Wee (5-50 mg kg(-1) , i.v.) on mean arterial pressure (MAP) and heart rate (HR) were determined in male Sprague-Dawley rats. The effects of L-W and L-Wee on basal tone and of phenylephrine- or KCl-induced contractions of aortic and MA rings were assessed. Effects of L-Wee and L-W on voltage-operated calcium channels (VOCC) of VSMC of MA were also examined in patch-clamp studies.

Key results: Administration of L-Wee, but not L-W, evoked a rapid and transient dose-dependent decrease in MAP and HR. While both agents failed to affect basal tone, L-Wee decreased, concentration-dependently, (I(max) > 98%) tension responses to phenylephrine and KCl in an endothelium-independent manner in aorta (IC(50) 2 mM) and MA (IC(50) 17 µM). L-Wee evoked concentration-dependent inhibition of VOCC currents (IC(50) 12 µM; I(max) 90%) in VSMC of MA.

Conclusions and implications: Esterified L-W (L-Wee), but not L-W, preferentially relaxed resistance vessels rather than conduit vessels. These effects were associated with blockade of VOCC by L-Wee. Our findings suggest that the falls in MAP and HR induced by L-Wee were due to blockade of VOCC by L-Wee.

Figure 1

The effects of acute i.v injection of increasing doses of either L-W (5–50 mg kg⁻¹, A) or and ethyl ester of L-W (L-Wee 5–50 mg·kg⁻¹, B) on changes in blood pressure and heart rate (HR–BPM) in 13 week-old male Sprague–Dawley rats, performed in parallel are shown. The changes in HR values determined as beats per minute (BPM) following administration of increasing doses of either L-W or L-Wee are also shown in A and B. The graphs show the mean (± SEM) data (n= 5 rats) for the % fall in mean arterial pressure (MAP, mmHg) or HR (BPM) attained following administration of indicated doses of L-Wee (C). **P < 0.01, significantly different from basal value before the addition of indicated dose of L-Wee.

Figure 2

Experimental records showing the lack of changes in the steady-state tonic response to cumulative addition of increasing concentrations of L-W (1 µM–8 mM) in endothelium-denuded rat aortic rings that were constricted by a fixed concentration of either the α₁-adrenoceptor selective agonist, phenylephrine (PE; 1 µM; A), or KCl (100 mM; B). The responses to addition of L-Wee (1 µM–8 mM) in parallel endothelial-denuded rings that were constricted with either phenylephrine (1 µM, C) or KCl (100 mM, D) are shown. Similar patterns of responses were obtained in aortic rings isolated from nine rats.

Figure 3

Experimental records showing the lack of changes in the sustained tonic response to cumulative addition of increasing concentrations of L-W (0.1 µM–640 µM) in endothelium-denuded third-order branches of rat superior MA that were constricted by either phenylephrine (PE; 10 µM; A) or KCl (100 mM, B). The responses to addition of L-Wee (0.1 µM–640 µM) in a parallel endothelial-denuded MA constricted with either phenylephrine (10 µM, C) or KCl (100 mM, D) are shown. Similar response patterns were obtained in vessels isolated from eight rats.

Figure 4

The comparison of concentration–inhibition response curves to L-W and L-Wee in endothelium-denuded aortic rings (A, B) and MA (C, D) that were constricted with a fixed concentration of either phenylephrine (PE; 1 or 10 µM) or KCl (100 mM) after their isolation from 13 week-old male Sprague–Dawley rats. Each data point is mean ± SEM value obtained from vessels isolated from a minimum of eight rats (n> 8 rats).

Figure 5

(A) Ba²⁺ currents recorded from a single VSMC before and after L-W (100 µM). Currents were elicited with voltage steps from −60 to +50 mV at time points as indicated in the time course (recorded at 0 mV) in lower panel. (B) Ba²⁺ currents recording before and after applications of L-Wee (100 µM) or nifedipine (5 µM). Lower panel shows the time course of currents at 0 mV. (C) Upper panel shows current–voltage relationships of nifedipine-sensitive currents in a VSMC before and after L-Wee (100 µM) application. Lower panel shows the current–voltage relationship of L-Wee sensitive currents (difference between currents before and after L-Wee treatment, n= 6). (D) Summary of mean ± SEM Ba²⁺ current density data in the presence of L-Wee at indicated concentrations (n= 7 VSMC from four rats). **P < 0.01, significantly different from 0 µM.