Vasostatin I (CgA17–76) vasoconstricts rat splanchnic vascular bed but does not affect central cardiovascular function

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Abstract

Vasostatin I (CgA1–76) is a naturally occurring biologically active peptide derived from chromogranin A (CgA), and is so named for its inhibitory effects on vascular tension. CgA mRNA is expressed abundantly in sympathoexcitatory catecholaminergic neurons of the rostral ventrolateral medulla (RVLM). CgA microinjection into the RVLM decreases blood pressure (BP), heart rate (HR) and sympathetic nerve activity (SNA). Proteolytic fragments of CgA are thought to be responsible for the cardiovascular effects observed. We hypothesised that vasostatin I is one of the fragments responsible for the central effects of CgA. We examined the role of a vasostatin I fragment, CgA17–76 (VS-I(CgA17–76)), containing the portion important for biological effects. The effects of VS-I(CgA17–76) delivered by intrathecal injection, or microinjection into the RVLM, on cardio-respiratory function in urethane anaesthetised, vagotomised, mechanically ventilated Sprague-Dawley rats (n = 21) were evaluated. The effects of intrathecal VS-I(CgA17–76) on the somato-sympathetic, baroreceptor and peripheral chemoreceptor reflexes were also examined. At the concentrations used (10, 100 or 200 μM, intrathecal; or 5 μM, RVLM microinjection) VS-I(CgA17–76) produced no change in mean arterial pressure, HR, splanchnic SNA, phrenic nerve amplitude or phrenic nerve frequency. All reflexes examined were unchanged following intrathecal VS-I(CgA17–76). In the periphery, VS-I(CgA17–76) potentiated the contractile effects of noradrenaline on rat mesenteric arteries (n = 6), with a significant left-shift in the dose response curve to noradrenaline (3.7 × 10−7 vs 7.7 × 10−7). Our results indicate that VS-I(CgA17–76) is active in the periphery but not centrally, and is not a central modulator of cardiorespiratory function and physiological reflexes.

Introduction

Chromogranin A (CgA) is a 439 amino acid protein that was first identified in the secretory granules of chromaffin cells of bovine adrenal medulla (Banks and Helle, 1965), but is now known to be distributed in other neuroendocrine tissues, including adrenergic and noradrenergic neurons of the central nervous system (Somogyi et al., 1984, Woulfe et al., 1999). CgA is co-stored with an assortment of neuromodulators and hormones including amines and peptides and is co-released by exocytosis. Mice lacking the CgA gene are hypertensive with increased left ventricular mass, suggesting an important role for CgA in autonomic control of circulation (Mahapatra et al., 2005).

Tissue- and species- specific proteolytic processing of CgA produces several biologically active peptides including catestatin (CgA344–364), vasostatin I (CgA1–76), vasostatin II (CgA1–113), and a variety of shorter vasostatin peptide fragments: CgA1–40, CgA7–57, CgA47–66, CgA67–76, that are found endogenously (Metz-Boutigue et al., 1993, Tota et al., 2008). Vasostatin I fragment (CgA17–39) is found as a circulating peptide in mammals (Stridsberg et al., 2000) and can produce peripheral cardiovascular effects, including suppression of blood vessel contraction (Aardal and Helle, 1992, Aardal et al., 1993, Brekke et al., 2002). Recently both vasostatin I and catestatin were reported to induce a negative inotropic effect on the isolated rat heart under basal conditions and after β-adrenergic stimulation (Cerra et al., 2006, Angelone et al., 2008).

While CgA peptide products clearly have peripheral cardiovascular effects, the function of these peptides within the central nervous system is less clear. Recently we reported that CgA mRNA is expressed in sympathoexcitatory catecholaminergic neurons within the rostral ventrolateral medulla (RVLM), a region important in establishment and maintenance of basal sympathetic tone (Gaede et al., 2009). In addition, catestatin was also found in catecholaminergic RVLM neurons and microinjection of catestatin into RVLM increased both mean arterial pressure (MAP) and sympathetic nerve activity (SNA), as well as modulated a number of cardiorespiratory reflexes (Gaede and Pilowsky, 2010). In contrast, intrathecal catestatin had no effect on cardiovascular parameters (Gaede et al., 2009). However, the effects of other CgA cleavage products, including vasostatin remain unknown. We aimed to examine the effects of a vasostatin I fragment CgA17–76 (VS-I(CgA17–76)) in the splanchnic vascular bed by in vitro myograph and on central cardiorespiratory function using intrathecal administration, or direct microinjection into the RVLM. This fragment of vasostatin I contains the disulfide-bridge region (CgA17–38SS) that is important for biological effects (Tota et al., 2003). We hypothesised that vasostatin I and catestatin (Gaede et al., 2009, Gaede and Pilowsky, 2010) may act through similar mechanisms and both peptides might exert similar cardiorespiratory actions when delivered centrally.

Section snippets

Experimental procedures

All animal experiments in this study complied with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (http://www.nhmrc.gov.au/publications/synopses/eal6syn.htm) and were approved by the Animal Ethics Committee of Macquarie University, Sydney, Australia.

Effects of intrathecal VS-I(CgA17–76)

The baseline MAP and heart rate (HR) in anaesthetized rats were 98 ± 5 mm Hg and 457 ± 12 bpm (n = 16). Intrathecal VS-I(CgA17–76) (10, 100 and 200 μM) produced no change in any of the cardiovascular parameters (MAP, HR or splanchnic SNA (sSNA)), or respiratory parameters (phrenic nerve amplitude (PNamp), phrenic nerve frequency (PNf)). A representative experiment is shown in Fig. 1A. Intrathecal injection of PBS caused no significant change in any of the above parameters. The magnitude of change in MAP,

Discussion

To the best of our knowledge, this is the first study to examine the central effect of VS-I(CgA17–76) on cardiorespiratory systems and reflexes and the effect of VS-I(CgA17–76) on rat mesenteric arteries. Our data demonstrate that i) VS-I(CgA17–76) applied in the CNS produces no cardiorespiratory effects under the conditions reported here, ii) intrathecal VS-I(CgA17–76) does not modulate sympathetic reflexes examined, and iii) VS-I(CgA17–76) enhances the effect of NA on rat mesenteric arteries.

Acknowledgments

Work in the Authors' laboratory is supported by grants from the National Health and Medical Research Council of Australia (457080, 457069), ARC (DP110102110), Macquarie University and the Garnett Passe and Rodney Williams Memorial Foundation. A.A.R. and I.Z.S. are recipients of Macquarie Research Excellence Scholarships. AMH is supported by a Coregas-Macquarie Research Excellence Scholarship. The authors have no conflicts of interest to declare.

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