Distribution of adrenomedullin-containing perivascular nerves in the rat mesenteric artery
Introduction
Adrenomedullin (AM) is a recently discovered peptide of 52 amino acid residues initially purified from an acid extract of human pheochromocytoma and showing potent hypotensive action when injected intravenously in rats [16]. AM has been detected in a variety of tissues, including the adrenal medulla, heart, kidney, lung, pancreas and brain in the humans and rats, but not in the peripheral nervous system [2], [8]. Cultured endothelial cells and vascular smooth muscle cells of different species have been shown to produce and secrete AM [29], [30]. Human AM mRNA was found to be highly expressed in several tissues including the adrenal medulla, heart (ventricle), lung and kidney as well as pheochromocytoma [17]. Also, AM mRNA was clearly detected in every region of the brain examined and in the pituitary [32]. AM-like immunoreactivity (AM-LI) was distributed in the brain region including the hypothalamus, paraventricular nucleus, Purkinje cells and perivascular process which may be derived from glial cells [28], suggesting neurotransmitter or neuromodulator functions of AM. However, there has been no report on AM distribution in the peripheral nervous system except in starfish [21].
AM has a partial sequence homology with calcitonin gene-related peptide (CGRP), which is a potent vasodilator peptide. The main distribution of CGRP in the peripheral site has been shown to be the primary sensory nerves [18], and the dorsal root ganglia (DRG) is a prominent site of CGRP synthesis and contains the cell bodies of the sensory afferent neurons [12], [20]. We have shown evidence that the rat mesenteric artery is innervated by nonadrenergic noncholinergic (NANC) vasodilator nerves in which CGRP acts as a neurotransmitter [5], [6], [14], [15]. Furthermore, we suggested that CGRP-containing nerves together with sympathetic adrenergic nerves regulate the tone of the blood vessels [13]. AM has been shown to act on CGRP receptors to produce vasodilation of the rat mesenteric artery [10], [24], [27]. This implies that AM may act as a neurotransmitter or neuromodulator in the mesenteric artery.
Therefore, the present study investigated whether AM is distributed in the perivascular nerves of the rat mesenteric artery using confocal laser and electron microscopic immunohistochemistry.
Section snippets
Animals
Male Wistar rats (Nippon Charles River, Shizuoka, Japan), weighing 280–300 g, were used in this study. The animals were given food and water at libitum, housed in the Animal Research Center of Okayama University at a controlled ambient temperature of 22±2 °C with 50±10% relative humidity and maintained on a 12:12 h light/dark cycle (light on at 8.00 a.m.).
Tissue processing
Animals were anesthetized with intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg), and then the mesenteric artery and DRG were
Results
As shown in Fig. 1A, a whole mount preparation of the rat mesenteric artery shows dense AM-like immunoreactive (AM-LI)-containing fibers, which surround the artery with reticular formation. In cross section of the mesenteric artery, AM-LI fibers were observed in the adventitial layer, but AM-LI fibers were also found in the tunica media and tunica intima (Fig. 1B). Preabsorption with rat-CGRP did not alter the immunostaining for AM-LI (Fig. 2A), while preabsorption with rat-AM (1–50) eliminated
Discussion
The present immunohistochemical study demonstrated that AM-LI-containing fibers were present in the mesenteric artery of the rat. The complete disappearance of AM-LI-containing fibers after cold storage denervation of the mesenteric artery as well as NPY-LI and CGRP-LI-containing fibers suggests that AM-LI-containing fibers are perivascular nerves and the nerves with AM may have some role in the perivascular neuronal function. Furthermore, abolishment of both AM-LI and CGRP-LI-containing fibers
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