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Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery
Life Sciences 88 (2011) 400–405
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Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery Feras Alkayed, Takahiko Shiina, Tadashi Takewaki, Yasutake Shimizu ⁎ Department of Basic Veterinary Science, Laboratory of Physiology, The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2010 Accepted 6 December 2010 Available online 16 December 2010 Keywords: Cholinergic innervation Excitatory neuromuscular transmission Purinergic innervation
a b s t r a c t Aims: The anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle. Main methods: Membrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). Key findings: Histological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens. Significance: Excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle. © 2010 Elsevier Inc. All rights reserved.
Introduction In mammals, vascular smooth muscle is oriented circularly and helically, while longitudinally orientated muscle is rare (Furchgott, 1955). However, the chicken anterior mesenteric artery contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer (Bennett and Malfmors, 1970). The longitudinal smooth muscle receives distinctive neural regulations. In the case of the circular smooth muscle in the chicken mesenteric artery, adrenergic stimulation produces contraction (Bell, 1969; Gooden, 1980), consistent with the fact that most mammalian arterial muscles receive excitatory adrenergic regulation (Keatinge, 1966). In contrast, the longitudinal smooth muscle is relaxed by adrenergic stimulation (Bolton, 1968, 1969). Moreover, the longitudinal muscle layer receives cholinergic innervation (Bell, 1969), and stimulation of cholinergic nerve fibers causes the muscle to contract (Bolton, 1968). In addition, there is an apparent difference in non-adrenergic non-
⁎ Corresponding author. Tel./fax: +81 58 293 2940. E-mail address: [email protected] (Y. Shimizu). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.12.012
cholinergic (NANC) regulation between longitudinal and circular smooth muscles. We have demonstrated that ATP released from perivascular nerves induces depolarization in longitudinal smooth muscles (Khalifa et al., 2005), whereas it causes hyperpolarization in circular smooth muscles through an endothelium-dependent mechanism (Draid et al., 2005). In general, development of innervation of most blood vessels consists of a gradual increase during pre- and postnatal stages with some decline in old age (Burnstock, 1975). At birth, no or little nerve terminal network is observed, whereas a plexus of nerve fibers and varicosities appear after the first postnatal week in several blood vessels of various mammalian species (Luff, 1999). In accordance with this, perivascular nerve stimulation induced contractile responses after the first postnatal week in rat portal veins (Ljung and Stage, 1975) and in rat irideal arterioles (Sandow and Hill, 1999). It has been shown that cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle of the anterior mesenteric artery of adult chickens (Bolton, 1968, 1969; Bell, 1969; Khalifa et al., 2005). However, there have been few reports on postnatal development of innervation to the muscles. In this study, we investigated cholinergic and purinergic regulation of
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longitudinal smooth muscle in the chicken anterior mesenteric artery in the neonatal period to clarify the developmental change in excitatory neurotransmission. Materials and methods Ethical approval This study was approved by the Gifu University Animal Care and Use Committee. This investigation was conducted in accordance with the Declaration of Helsinki. All efforts were made to minimize animal suffering and to reduce the number of animals used. Animals and tissue preparation Healthy male white leghorn chickens aged 1–10 days and some aged 12 weeks were used in the experiments. The chickens were killed by neck dislocation. A segment of the anterior mesenteric artery was cut at its point of origin from the aorta and placed in physiological salt solution (PSS) at room temperature. After removing connective tissue, the proximal end portion was cannulated with a glass micropipette attached to a gravity-driven perfusion apparatus to remove blood by perfusing with warmed (29 °C) PSS. Electrophysiological recordings Arteries were placed in a partition chamber in which large extracellular silver-silver chloride plates were placed to elicit nerve stimulation, as described previously (Khalifa et al., 2005). Each preparation was perfused with warmed (29 °C) PSS at a constant flow rate (3 mL/min). The temperature of 29 °C was used in this experiment since spontaneous activity, which makes recording from the cells difficult, was frequently produced at 35 °C (Khalifa et al., 2005). Tissue preparations were subsequently allowed to equilibrate for 1 h. Microelectrode insertions were made in longitudinal muscle cells through the adventitial side, within 2 mm of the stimulation electrode. Membrane potentials were recorded with a conventional microelectrode technique using a glass capillary microelectrode filled with 3 M KCl with tip resistances ranging from 50 to 80 MΩ (Takewaki and Ohashi, 1977). Electrical activity was monitored on an oscilloscope (CS 4026, Kenwood, Tokyo, Japan) and recorded using the PowerLab system (ADInstruments, Bella Vista NSW, Australia). The perivascular nerves were stimulated by applying electrical field stimulation (EFS; stimulus intensity: 15 V, pulse duration: 1 ms, frequency: 20 Hz) for 0.5-s periods.
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The drugs used were as follows: ACh, ATP, atropine sulfate monohydrate, suramin sodium, and tetrodotoxin (TTX) (Sigma, St Louis, MO, USA). All drugs were dissolved in distilled water (1000 times or higher concentrations) and were further diluted by PSS just before use. ACh and ATP were applied by focal application, whereas other drugs were applied at required concentrations by adding to superfusing PSS. Histological examination The portion of the middle artery of 1-day-old chickens was separated. Then the dissected artery was fixed in Bouin's solution (saturated picric acid/formalin/acetic acid = 15/5/1) for 16 h at 4 °C, sectioned at 10 μm in thickness, and stained with hematoxylin and eosin. RNA isolation and reverse transcriptase-PCR (RT-PCR) The expression of purinoceptor gene mRNA was assessed by RTPCR. Total cellular RNA was extracted from tissue homogenates using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 2 mg of total RNA by using Superscript III RNase Hreverse transcriptase (Invitrogen) and Random primers (Invitrogen). The absence of PCR-amplified DNA fragments in the samples without reverse transcription indicated the isolation of RNA free from genomic DNA contamination. The PCR was performed with platinum Taq DNA polymerase (Invitrogen). The primer sets were as follows: P2X1 receptor sense 5′-ACT GGT GCG GAA CAA GAA GG-3′ and anti-sense 5′-TCA CAG TTC CAG TCG ATG GT-3′ (predicted size = 720 bp); P2X4 receptor sense 5′-ACA GAT TCC GTG GTC AGT TC-3′ and anti-sense 5′CAA ATG TTG TTC TTT ACC AG-3′ (predicted size = 413 bp); P2X5 receptor sense 5′-GTG TCC TGG AAG GGT TTA T-3′ and anti-sense 5′GAA GTG GTT TCT TGG GCT TG-3′ (predicted size = 535 bp); P2Y1 receptor sense 5′-CAT CTC TGC TGC TCT GAA CG-3′ and anti-sense 5′TGG TTT TGT TCC TCC TCA CC-3′ (predicted size = 551 bp); P2Y5 receptor sense 5′-TAA GCT CTA ACT GCT CCA CT-3′ and anti-sense 5′TCA GGG TAA TGT TAT AAG GC-3′ (predicted size = 735 bp); P2Y6 receptor sense 5′-TGG TCT ACT CAG TGG TGT TC-3′ and anti-sense 5′AGT GAT GGT CAA CGT GAT GC-3′ (predicted size = 506 bp); β-actin sense 5′-TGA CCC TGA AGT ACC CCA TTG-3′ and anti-sense 5′-TCA GGA TCT TCA TGA GGT AG-3′ (predicted size = 387 bp). All primers were purchased from Invitrogen. Amplifications were performed by 30 cycles. The reaction products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide (0.4 μg/mL). The gels were exposed to UV light with a UV transilluminator (UVP Laboratory Products, Upland, CA, USA).
Focal application system Data analysis A pneumatic pump was used for the application of measured quantities of acetylcholine (ACh) or ATP to localized regions and at a particular time. Fiber-filled glass micropipettes (outside diameter = 1 mm, inside diameter = 0.5 mm) were drawn with a microelectrode puller (Narishige, Japan, type PP-83), yielding an outside diameter of the tip of 20–30 μm. The pipette was then filled with a solution of ACh or ATP. Drugs were pressure-ejected from a micropipette with a pressure of 10 psi and pulse duration of 10 ms. After application of the drug, the pipette was withdrawn.
Experimental data obtained are presented as means ± standard deviation (SD). n indicates the number of separate arteries in which electrical events were recorded. Statistical analysis was performed with Student's unpaired t-test, and P-values b 0.05 were considered to be statistically significant. Results
Drugs and solutions
Histological examination of the anterior mesenteric artery in 1-day-old chickens
The PSS used in this study had the following composition in mM: NaCl 118, KCl 4.6, CaCl2 2.7, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11. The solution in the supply reservoir was gassed continuously with 95% O2:5% CO2 gas mixture creating a pH of 7.2 and was warmed to 29 °C.
The existence of a longitudinal smooth muscle layer in the anterior mesenteric artery of 1-day-old chickens was investigated. As shown in Fig. 1, longitudinal smooth muscles were found in adventitia in addition to circular smooth muscles in arterial media. The thickness ratio of the longitudinal smooth muscle layer to circular layer was
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Fig. 1. Anterior mesenteric artery of a 1-day-old chicken stained with hematoxylin and eosin. A cross section of the middle anterior mesenteric artery shows the existence of a longitudinal smooth muscle layer in adventitia in addition to circular smooth muscle in arterial media. LM: longitudinal smooth muscle (17–21 μm in diameter), CM: circular smooth muscle (25–30 μm in diameter).
0.72 ± 0.04, which is significantly lower than the ratio of adult chickens (0.90 ± 0.07).
Effects of perivascular nerve stimulation on membrane potential of longitudinal smooth muscle in the anterior mesenteric artery of 1-dayold chickens The mean resting membrane potential from longitudinal smooth muscle cells of the anterior mesenteric artery in 1-day-old chickens was −33.8 ± 3.1 mV (n = 15) and was not significantly different from that in 12-week-old chickens (−35.7 ± 3.1 mV, n = 10). To investigate neuronal control of longitudinal smooth muscles in 1-day-old chickens, the perivascular nerves were stimulated by EFS and membrane response of the smooth muscle was recorded. In agreement with our previous study (Alkayed et al., 2009), EFS (15 V, 1 ms, 20 Hz for 0.5 s) induced a depolarization response, i.e. excitatory junction potential (EJP), in 12-week-old chickens (Fig. 2B and C). The EJP was blocked by 0.3 μM of TTX (data not shown), indicating that the response was neurogenic. In contrast, EFS under the same conditions did not elicit any responses in artery preparations isolated from 1-day-old chicken (Fig. 2A and C). Employing the multiple pulse stimulation at 20 Hz also had no effect on membrane potential of longitudinal smooth muscle cells (data not shown).
Fig. 2. Effects of electrical field stimulation (EFS) on membrane responses of longitudinal smooth muscle of the chicken anterior mesenteric artery. Typical recordings of EFS-evoked responses in 1-day-old (A) and 12-week-old (B) chickens are shown. Resting membrane potentials for A and B were −33 mV and −35 mV, respectively. Similar results were obtained in six independent experiments. (C) The EFS-evoked EJPs in 1-day-old and 12-week-old chickens are summarized (n = 6). Each bar represents the mean of data ± S.D.
Age-dependent changes in EFS-evoked response in longitudinal smooth muscle of the chicken anterior mesenteric artery Since the post-synaptic machinery that promotes membrane responses would be functional in longitudinal smooth muscle of the chicken anterior mesenteric artery at 1 day of age, we examined agedependent changes in membrane responses to perivascular nerve stimulation. EFS induced a small EJP in longitudinal smooth muscle of
Effect of exogenous application of ACh and ATP on membrane potential of longitudinal smooth muscle in 1-day-old chickens To clarify whether the absence of EFS-induced response in the longitudinal smooth muscle of 1-day-old chickens is due to rudimentary innervation or due to a defect of post-synaptic machinery, we exogenously applied ACh or ATP instead of EFS. As shown in Fig. 3A, exogenous application of ACh (5 μM) produced a slow depolarization. The ACh-induced depolarization was abolished by pretreatment with a muscarinic cholinoceptor blocker, atropine (0.5 μM). ATP at a final concentration of 5 μM also produced depolarization in longitudinal smooth muscle of the anterior mesenteric artery isolated from 1-day-old chickens. ATP failed to induce depolarization in the presence of a non-specific purinoceptor antagonist, suramin (50 μM) (Fig. 3B). Similar agonist-induced depolarizations were observed in the cells isolated from 5-day-old and 10-day-old chickens, suggesting that cellular responsiveness is not changed during early postnatal period.
Fig. 3. Effects of exogenous application of ACh and ATP on membrane potential of longitudinal smooth muscles in 1-day-old chickens. Typical recordings after exogenous application of ACh (5 μM; A) in the absence (control) or presence of atropine (0.5 μM) and ATP (5 μM; B) in the absence (control) or presence of suramin (50 μM) are shown. Resting membrane potentials for A and B were −32 mV and −35 mV, respectively. Similar results were obtained in five independent experiments.
F. Alkayed et al. / Life Sciences 88 (2011) 400–405
Fig. 4. Age-dependent changes in EFS-evoked response in longitudinal smooth muscles of the chicken anterior mesenteric artery. EFS evoked slow EJP in 5-day-old chickens (A) and more prominent EJP in 10-day-old chickens (B). Resting membrane potentials for A and B were −31 mV and −34 mV, respectively. Similar results were obtained in five independent experiments. (C) The EFS-evoked EJPs in 5-day-old and 10-day-old chickens are summarized (n = 5). Each bar represents the mean of data ± S.D.
5-day-old chickens (Fig. 4A and C). At 10 days of age, the EJP was more prominent (Fig. 4B and C) but was still less than that observed in 12week-old chickens (see Fig. 2B and C). Pretreatment of tissue preparation obtained from 10-day-old chickens with a muscarinic cholinoceptor blocker, atropine (0.5 μM), completely abolished the EJP (Fig. 5). Atropine did not
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Fig. 6. Effects of suramin on EJP evoked by EFS. (A) Typical recordings of EFS-evoked responses in longitudinal smooth muscle of the anterior mesenteric artery of 10-dayold chickens in the absence (control) or presence of suramin (500 μM) are shown. Similar results were obtained in five independent experiments. (B) The effects of suramin (500 μM) on the EFS-evoked EJP are summarized (n = 5). Each bar represents the mean of data ± S.D.
significantly affect resting membrane potential (−33.3 ± 1.2 mV, n = 10). In contrast, a non-specific purinoceptor antagonist, suramin (500 μM), had no effect on the EFS-evoked EJP (Fig. 6). Molecular identification of purinoceptors in the chicken anterior mesenteric artery by RT-PCR We then examined expression of purinoceptor isoforms (nomeculture following Alexander et al. 2008) in the anterior mesenteric artery of 10-day-old chickens by using RT-PCR. Amplified products of P2X1, P2X4, P2X5, P2Y1, P2Y5 and P2Y6 were observed in appropriate sizes (Fig. 7A). Similar expression pattern of purinoceptor isoforms were observed in the anterior mesenteric artery of 12-week-old chickens (Fig. 7B). Discussion
Fig. 5. Effects of atropine on EJP evoked by EFS. (A) Typical recordings of EFS-evoked responses in longitudinal smooth muscle of the anterior mesenteric artery of 10-dayold chickens in the absence (control) or presence of atropine (0.5 μM) are shown. Similar results were obtained in five independent experiments. (B) The inhibitory effects of atropine (0.5 μM) on the EFS-evoked EJP are summarized (n = 5). Each bar represents the mean of data ± S.D. *p b 0.05, compared to the control.
We previously showed that perivascular nerve stimulation evoked EJP of longitudinal smooth muscles in anterior mesenteric arteries of adult chickens (Khalifa et al., 2005). In the present study, we investigated postnatal development of excitatory innervation to longitudinal smooth muscles. The major findings are (i) a welldeveloped longitudinal smooth muscle layer exists in the anterior mesenteric artery of 1-day-old chickens, (ii) perivascular nerve stimulation failed to evoke response of membrane potential in the smooth muscle of 1-day-old chickens, although it caused a large EJP in 12-week-old chickens, (iii) the smooth muscle of 1-day-old chickens showed depolarization in response to exogenously applied ACh or ATP, (iv) EFS-evoked EJP was observed in 5-day-old chickens and became more prominent at the age of 10 days, (v) the EJP in 10-dayold chickens was blocked by a muscarinic ACh receptor blocker (atropine), whereas blockade of purinoceptors by a non-specific purinoceptor antagonist, suramin, had no effect, and (vi) several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in the smooth muscle of adult chicken (Khalifa et al., 2005), were expressed in the anterior mesenteric artery of 10day-old chickens. These results demonstrate that excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric
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Fig. 7. Identification of purinoceptor isoforms in the chicken anterior mesenteric artery by RT-PCR. Homogenized samples from the anterior mesenteric artery of 10-day-old (A) and 12-week-old (B) chickens were used for RT-PCR. Amplified products of P2X1, P2X4, P2X5, P2Y1, P2Y5 and P2Y6 purinoceptors were observed in appropriate sizes in the artery.
artery is not established at birth but developed during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in the smooth muscle. In the rat portal vein, the longitudinal smooth muscle layer is not found at birth (Ts'Ao et al., 1971) but becomes apparent at 2 weeks of age (Lundberg et al., 1976). Taking into consideration that the longitudinal smooth muscle layer develops after birth in the vein, it was of interest to determine whether the chicken anterior mesenteric artery possesses a longitudinal smooth muscle layer at birth. Hematoxylin and eosin staining of cross-sections of the artery revealed that a well-developed longitudinal smooth muscle layer exists at 1 day of age. To our knowledge, this is the first demonstration of the presence of an outer longitudinal muscle layer in the artery of a newly hatched chicken. Perivascular nerve stimulation failed to produce any responses in the longitudinal smooth muscle of 1-day-old chickens. The same stimulus protocol substantially induced EJP in preparations isolated from 12-week-old chickens. It thus seems likely that the EFS applied to the preparations is sufficient to stimulate perivascular nerves. In accordance with this, multiple pulse stimulation at 20 Hz was still insufficient to evoke membrane response in 1-day-old chickens. One possible explanation for the failure of EFS to induce membrane response is a defect in responsiveness of the smooth muscle to excitatory neurotransmitters, including ACh and ATP. However, this is not the case, since exogenous application of ACh or ATP could induce depolarization even in preparations obtained from 1-day-old chickens (Fig. 3). Alternatively, it is reasonable to assume that excitatory neuromuscular transmission is not completed and/or effective innervations are not well-established in newly hatched chickens. This assumption is consistent with results of a study by Ljung and
Stage (1975), showing that responsiveness of vascular smooth muscle to exogenous noradrenaline is established before the development of sympathetic innervation. Although perivascular nerve stimulation failed to elicit EJP of the longitudinal smooth muscle in newly hatched chickens, it was capable of eliciting a slight EJP in 5-day-old chickens (Fig. 4A). As preparation obtained from 10-day-old chicken showed more prominent EJP in response to perivascular nerve stimulation (Fig. 4B), it can be considered that excitatory neuromuscular transmission gradually develops during the early postnatal period. It was previously demonstrated that both cholinergic and purinergic components are involved in depolarizing response of the longitudinal smooth muscle in mature chickens (Khalifa et al., 2005). However, a non-specific purinoceptor antagonist, suramin, had no effect on the depolarizing response of 10-day-old chickens (Fig. 6), although purinoceptors found in the anterior mesenteric artery of adult chickens were already expressed in the artery at the age (Fig. 7). In addition, the EJP was completely abolished by a muscarinic ACh receptor antagonist, atropine (Fig. 5), while the antagonist only partially attenuated EJP observed in mature chickens (Khalifa et al., 2005). These results suggest that cholinergic nerves exclusively innervate the longitudinal smooth muscle in the early postnatal period. Thus, it is concluded that each nerve component does not develop uniformly; cholinergic innervation, which begins as early as 5 days of age, precedes purinergic innervation. At present, the timing of purinergic innervation is unclear. In relation with this, however, our previous study showed that purinergic excitatory neuromuscular transmission is still not obvious in chicken aged 5 weeks (Alkayed et al., 2009). Recently, we have demonstrated that activation of perivascular purinergic component in relatively young chickens (5 weeks of age) causes prominent hyperpolarization, not depolarization, in longitudinal smooth muscle of the anterior mesenteric artery (Alkayed et al., 2009). The events associated with the hyperpolarization consist of ATP release from nerve terminals, its binding to endothelial P2X purinoceptors, generation of nitric oxide (NO) in endothelial cells, diffusion of NO to the longitudinal smooth muscle, and activation of guanylate cyclase. The present results also suggest that the inhibitory purinergic component is not established in newly hatched chickens. Together with our previous findings (Alkayed et al., 2009; Khalifa et al., 2005), our data provide an overview of developmental changes in neural components that control the longitudinal smooth muscle of the chicken anterior mesenteric artery as summarized in Table 1. The physiological significance of the age-dependent shift of purinergic regulation, i.e., shift from inhibitory to excitatory, is unclear at present. The longitudinal smooth muscle would play a minor role, if any, in regulation of vascular tone, since contraction of longitudinal smooth muscle had little effect on perfusion pressure in the isolated chicken mesenteric artery (Bell, 1969). In the case of the posterior mesenteric artery of chickens, the longitudinal muscle layer was found only in females (unpublished observation). Remarkably, occurrence of the longitudinal muscle layer in female chickens became obvious when chickens began egg production. Therefore, it is reasonable to assume that the longitudinal smooth muscle confers resistance to tension applied to blood vessels. Based on this assumption, the age-dependent shift of purinergic regulation can be explained as an adaptive change against putative higher tension
Table 1 Developmental changes in neuronal components of the chicken anterior mesenteric artery. Neuronal control
1 day
10 days
5 weeks
12 weeks
Excitatory cholinergic component Excitatory purinergic component Inhibitory purinergic component (Endothelium-dependent)
− − −
± − −
+ − +
+ + ±
F. Alkayed et al. / Life Sciences 88 (2011) 400–405
applied to the anterior mesenteric artery in mature chickens. Further study is needed to elucidate the physiological roles of the longitudinal smooth muscle and developmental changes in regulatory neural components. Conclusion The present study suggests that excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but developed during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in the smooth muscle. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgment This research was supported by a Grant-in-Aid Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Alexander SPH, Mathie A, Peters JA. Guide to receptors and channels (GRAC), 3rd edn. Br J Pharmacol 2008;153 (Suppl. 2): S1–S209. Alkayed F, Boudaka A, Shiina T, Takewaki T, Shimizu Y. P2X purinoceptors mediate an endothelium-dependent hyperpolarization in longitudinal smooth muscle of anterior mesenteric artery in young chickens. Br J Pharmacol 2009;158(3):888–95. Bell C. Indirect cholinergic vasomotor control of intestinal blood flow in the domestic chicken. J Physiol 1969;205(2):317–27.
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Bennett T, Malfmors T. The adrenergic nervous system of the domestic fowl (Gallus domesticus (L.)). Z Zellforsch Mikrosk Anat 1970;106(1):22–50. Bolton TB. Studies on the longitudinal muscle of the anterior mesenteric artery of the domestic fowl. J Physiol 1968;196(2):273–81. Bolton TB. Spontaneous and evoked release of neurotransmitter substances in the longitudinal muscle of anterior mesenteric artery of the domestic fowl. Br J Pharmacol 1969;35(1):112–20. Burnstock G. Innervation of vascular smooth muscle: histochemistry and electron microscopy. Clin Exp Pharmacol Physiol Suppl 1975;2:7-20. Draid M, Shiina T, El-Mahmoudy A, Boudaka A, Shimizu Y, Takewaki T. Neurally released ATP mediates endothelium-dependent hyperpolarization in the circular smooth muscle cells of chicken anterior mesenteric artery. Br J Pharmacol 2005;146(7): 983–9. Furchgott RF. The pharmacology of vascular smooth muscle. Pharmacol Rev 1955;7(2): 183–265. Gooden BA. The effect of hypoxia on vasoconstrictor responses of isolated mesenteric arterial vasculature from chicken and duckling. Comp Biochem Physiol C Comp Pharmacol 1980;67(2):219–22. Keatinge WR. Electrical and mechanical response of arteries to stimulation of sympathetic nerves. J Physiol 1966;185(3):701–15. Khalifa M, El-mahmoudy A, Shiina T, Shimizu Y, Nikami H, El-Sayed M, Kobayashi H, Takewaki T. An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery. Br J Pharmacol 2005;144(6):830–9. Ljung B, Stage D. Postnatal ontogenetic development of neurogenic and myogenic control in the rat portal vein. Acta Physiol Scand 1975;94(1):112–27. Luff SE. Development of neuromuscular junctions on small mesenteric arteries of the rat. J Neurocytol 1999;28(1):47–62. Lundberg J, Ljung B, Stage (McMurphy) D, Dahlström A. Postnatal ontogenic development of the adrenergic innervation pattern in the rat portal vein. A histochemical study. Cell Tissue Res 1976;172(1):15–27. Sandow LS, Hill EC. Physiological and anatomical studies of the development of the sympathetic innervation to rat iris arterioles. J Auton Nerv Syst 1999;77(2–3): 152–63. Takewaki T, Ohashi O. Non-cholinergic excitatory transmission to intestinal smooth muscle cells. Nature 1977;268(5622):749–50. Ts'Ao CH, Glagov S, Kelsey BF. Structure of mammalian portal vein: postnatal establishment of two mutually perpendicular medial muscle zones in the rat. Anat Rec 1971;171(4):457–70.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery Feras Alkayed, Takahiko Shiina, Tadashi Takewaki, Yasutake Shimizu ⁎ Department of Basic Veterinary Science, Laboratory of Physiology, The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2010 Accepted 6 December 2010 Available online 16 December 2010 Keywords: Cholinergic innervation Excitatory neuromuscular transmission Purinergic innervation
a b s t r a c t Aims: The anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle. Main methods: Membrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). Key findings: Histological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens. Significance: Excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle. © 2010 Elsevier Inc. All rights reserved.
Introduction In mammals, vascular smooth muscle is oriented circularly and helically, while longitudinally orientated muscle is rare (Furchgott, 1955). However, the chicken anterior mesenteric artery contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer (Bennett and Malfmors, 1970). The longitudinal smooth muscle receives distinctive neural regulations. In the case of the circular smooth muscle in the chicken mesenteric artery, adrenergic stimulation produces contraction (Bell, 1969; Gooden, 1980), consistent with the fact that most mammalian arterial muscles receive excitatory adrenergic regulation (Keatinge, 1966). In contrast, the longitudinal smooth muscle is relaxed by adrenergic stimulation (Bolton, 1968, 1969). Moreover, the longitudinal muscle layer receives cholinergic innervation (Bell, 1969), and stimulation of cholinergic nerve fibers causes the muscle to contract (Bolton, 1968). In addition, there is an apparent difference in non-adrenergic non-
⁎ Corresponding author. Tel./fax: +81 58 293 2940. E-mail address: [email protected] (Y. Shimizu). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.12.012
cholinergic (NANC) regulation between longitudinal and circular smooth muscles. We have demonstrated that ATP released from perivascular nerves induces depolarization in longitudinal smooth muscles (Khalifa et al., 2005), whereas it causes hyperpolarization in circular smooth muscles through an endothelium-dependent mechanism (Draid et al., 2005). In general, development of innervation of most blood vessels consists of a gradual increase during pre- and postnatal stages with some decline in old age (Burnstock, 1975). At birth, no or little nerve terminal network is observed, whereas a plexus of nerve fibers and varicosities appear after the first postnatal week in several blood vessels of various mammalian species (Luff, 1999). In accordance with this, perivascular nerve stimulation induced contractile responses after the first postnatal week in rat portal veins (Ljung and Stage, 1975) and in rat irideal arterioles (Sandow and Hill, 1999). It has been shown that cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle of the anterior mesenteric artery of adult chickens (Bolton, 1968, 1969; Bell, 1969; Khalifa et al., 2005). However, there have been few reports on postnatal development of innervation to the muscles. In this study, we investigated cholinergic and purinergic regulation of
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longitudinal smooth muscle in the chicken anterior mesenteric artery in the neonatal period to clarify the developmental change in excitatory neurotransmission. Materials and methods Ethical approval This study was approved by the Gifu University Animal Care and Use Committee. This investigation was conducted in accordance with the Declaration of Helsinki. All efforts were made to minimize animal suffering and to reduce the number of animals used. Animals and tissue preparation Healthy male white leghorn chickens aged 1–10 days and some aged 12 weeks were used in the experiments. The chickens were killed by neck dislocation. A segment of the anterior mesenteric artery was cut at its point of origin from the aorta and placed in physiological salt solution (PSS) at room temperature. After removing connective tissue, the proximal end portion was cannulated with a glass micropipette attached to a gravity-driven perfusion apparatus to remove blood by perfusing with warmed (29 °C) PSS. Electrophysiological recordings Arteries were placed in a partition chamber in which large extracellular silver-silver chloride plates were placed to elicit nerve stimulation, as described previously (Khalifa et al., 2005). Each preparation was perfused with warmed (29 °C) PSS at a constant flow rate (3 mL/min). The temperature of 29 °C was used in this experiment since spontaneous activity, which makes recording from the cells difficult, was frequently produced at 35 °C (Khalifa et al., 2005). Tissue preparations were subsequently allowed to equilibrate for 1 h. Microelectrode insertions were made in longitudinal muscle cells through the adventitial side, within 2 mm of the stimulation electrode. Membrane potentials were recorded with a conventional microelectrode technique using a glass capillary microelectrode filled with 3 M KCl with tip resistances ranging from 50 to 80 MΩ (Takewaki and Ohashi, 1977). Electrical activity was monitored on an oscilloscope (CS 4026, Kenwood, Tokyo, Japan) and recorded using the PowerLab system (ADInstruments, Bella Vista NSW, Australia). The perivascular nerves were stimulated by applying electrical field stimulation (EFS; stimulus intensity: 15 V, pulse duration: 1 ms, frequency: 20 Hz) for 0.5-s periods.
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The drugs used were as follows: ACh, ATP, atropine sulfate monohydrate, suramin sodium, and tetrodotoxin (TTX) (Sigma, St Louis, MO, USA). All drugs were dissolved in distilled water (1000 times or higher concentrations) and were further diluted by PSS just before use. ACh and ATP were applied by focal application, whereas other drugs were applied at required concentrations by adding to superfusing PSS. Histological examination The portion of the middle artery of 1-day-old chickens was separated. Then the dissected artery was fixed in Bouin's solution (saturated picric acid/formalin/acetic acid = 15/5/1) for 16 h at 4 °C, sectioned at 10 μm in thickness, and stained with hematoxylin and eosin. RNA isolation and reverse transcriptase-PCR (RT-PCR) The expression of purinoceptor gene mRNA was assessed by RTPCR. Total cellular RNA was extracted from tissue homogenates using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 2 mg of total RNA by using Superscript III RNase Hreverse transcriptase (Invitrogen) and Random primers (Invitrogen). The absence of PCR-amplified DNA fragments in the samples without reverse transcription indicated the isolation of RNA free from genomic DNA contamination. The PCR was performed with platinum Taq DNA polymerase (Invitrogen). The primer sets were as follows: P2X1 receptor sense 5′-ACT GGT GCG GAA CAA GAA GG-3′ and anti-sense 5′-TCA CAG TTC CAG TCG ATG GT-3′ (predicted size = 720 bp); P2X4 receptor sense 5′-ACA GAT TCC GTG GTC AGT TC-3′ and anti-sense 5′CAA ATG TTG TTC TTT ACC AG-3′ (predicted size = 413 bp); P2X5 receptor sense 5′-GTG TCC TGG AAG GGT TTA T-3′ and anti-sense 5′GAA GTG GTT TCT TGG GCT TG-3′ (predicted size = 535 bp); P2Y1 receptor sense 5′-CAT CTC TGC TGC TCT GAA CG-3′ and anti-sense 5′TGG TTT TGT TCC TCC TCA CC-3′ (predicted size = 551 bp); P2Y5 receptor sense 5′-TAA GCT CTA ACT GCT CCA CT-3′ and anti-sense 5′TCA GGG TAA TGT TAT AAG GC-3′ (predicted size = 735 bp); P2Y6 receptor sense 5′-TGG TCT ACT CAG TGG TGT TC-3′ and anti-sense 5′AGT GAT GGT CAA CGT GAT GC-3′ (predicted size = 506 bp); β-actin sense 5′-TGA CCC TGA AGT ACC CCA TTG-3′ and anti-sense 5′-TCA GGA TCT TCA TGA GGT AG-3′ (predicted size = 387 bp). All primers were purchased from Invitrogen. Amplifications were performed by 30 cycles. The reaction products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide (0.4 μg/mL). The gels were exposed to UV light with a UV transilluminator (UVP Laboratory Products, Upland, CA, USA).
Focal application system Data analysis A pneumatic pump was used for the application of measured quantities of acetylcholine (ACh) or ATP to localized regions and at a particular time. Fiber-filled glass micropipettes (outside diameter = 1 mm, inside diameter = 0.5 mm) were drawn with a microelectrode puller (Narishige, Japan, type PP-83), yielding an outside diameter of the tip of 20–30 μm. The pipette was then filled with a solution of ACh or ATP. Drugs were pressure-ejected from a micropipette with a pressure of 10 psi and pulse duration of 10 ms. After application of the drug, the pipette was withdrawn.
Experimental data obtained are presented as means ± standard deviation (SD). n indicates the number of separate arteries in which electrical events were recorded. Statistical analysis was performed with Student's unpaired t-test, and P-values b 0.05 were considered to be statistically significant. Results
Drugs and solutions
Histological examination of the anterior mesenteric artery in 1-day-old chickens
The PSS used in this study had the following composition in mM: NaCl 118, KCl 4.6, CaCl2 2.7, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11. The solution in the supply reservoir was gassed continuously with 95% O2:5% CO2 gas mixture creating a pH of 7.2 and was warmed to 29 °C.
The existence of a longitudinal smooth muscle layer in the anterior mesenteric artery of 1-day-old chickens was investigated. As shown in Fig. 1, longitudinal smooth muscles were found in adventitia in addition to circular smooth muscles in arterial media. The thickness ratio of the longitudinal smooth muscle layer to circular layer was
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Fig. 1. Anterior mesenteric artery of a 1-day-old chicken stained with hematoxylin and eosin. A cross section of the middle anterior mesenteric artery shows the existence of a longitudinal smooth muscle layer in adventitia in addition to circular smooth muscle in arterial media. LM: longitudinal smooth muscle (17–21 μm in diameter), CM: circular smooth muscle (25–30 μm in diameter).
0.72 ± 0.04, which is significantly lower than the ratio of adult chickens (0.90 ± 0.07).
Effects of perivascular nerve stimulation on membrane potential of longitudinal smooth muscle in the anterior mesenteric artery of 1-dayold chickens The mean resting membrane potential from longitudinal smooth muscle cells of the anterior mesenteric artery in 1-day-old chickens was −33.8 ± 3.1 mV (n = 15) and was not significantly different from that in 12-week-old chickens (−35.7 ± 3.1 mV, n = 10). To investigate neuronal control of longitudinal smooth muscles in 1-day-old chickens, the perivascular nerves were stimulated by EFS and membrane response of the smooth muscle was recorded. In agreement with our previous study (Alkayed et al., 2009), EFS (15 V, 1 ms, 20 Hz for 0.5 s) induced a depolarization response, i.e. excitatory junction potential (EJP), in 12-week-old chickens (Fig. 2B and C). The EJP was blocked by 0.3 μM of TTX (data not shown), indicating that the response was neurogenic. In contrast, EFS under the same conditions did not elicit any responses in artery preparations isolated from 1-day-old chicken (Fig. 2A and C). Employing the multiple pulse stimulation at 20 Hz also had no effect on membrane potential of longitudinal smooth muscle cells (data not shown).
Fig. 2. Effects of electrical field stimulation (EFS) on membrane responses of longitudinal smooth muscle of the chicken anterior mesenteric artery. Typical recordings of EFS-evoked responses in 1-day-old (A) and 12-week-old (B) chickens are shown. Resting membrane potentials for A and B were −33 mV and −35 mV, respectively. Similar results were obtained in six independent experiments. (C) The EFS-evoked EJPs in 1-day-old and 12-week-old chickens are summarized (n = 6). Each bar represents the mean of data ± S.D.
Age-dependent changes in EFS-evoked response in longitudinal smooth muscle of the chicken anterior mesenteric artery Since the post-synaptic machinery that promotes membrane responses would be functional in longitudinal smooth muscle of the chicken anterior mesenteric artery at 1 day of age, we examined agedependent changes in membrane responses to perivascular nerve stimulation. EFS induced a small EJP in longitudinal smooth muscle of
Effect of exogenous application of ACh and ATP on membrane potential of longitudinal smooth muscle in 1-day-old chickens To clarify whether the absence of EFS-induced response in the longitudinal smooth muscle of 1-day-old chickens is due to rudimentary innervation or due to a defect of post-synaptic machinery, we exogenously applied ACh or ATP instead of EFS. As shown in Fig. 3A, exogenous application of ACh (5 μM) produced a slow depolarization. The ACh-induced depolarization was abolished by pretreatment with a muscarinic cholinoceptor blocker, atropine (0.5 μM). ATP at a final concentration of 5 μM also produced depolarization in longitudinal smooth muscle of the anterior mesenteric artery isolated from 1-day-old chickens. ATP failed to induce depolarization in the presence of a non-specific purinoceptor antagonist, suramin (50 μM) (Fig. 3B). Similar agonist-induced depolarizations were observed in the cells isolated from 5-day-old and 10-day-old chickens, suggesting that cellular responsiveness is not changed during early postnatal period.
Fig. 3. Effects of exogenous application of ACh and ATP on membrane potential of longitudinal smooth muscles in 1-day-old chickens. Typical recordings after exogenous application of ACh (5 μM; A) in the absence (control) or presence of atropine (0.5 μM) and ATP (5 μM; B) in the absence (control) or presence of suramin (50 μM) are shown. Resting membrane potentials for A and B were −32 mV and −35 mV, respectively. Similar results were obtained in five independent experiments.
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Fig. 4. Age-dependent changes in EFS-evoked response in longitudinal smooth muscles of the chicken anterior mesenteric artery. EFS evoked slow EJP in 5-day-old chickens (A) and more prominent EJP in 10-day-old chickens (B). Resting membrane potentials for A and B were −31 mV and −34 mV, respectively. Similar results were obtained in five independent experiments. (C) The EFS-evoked EJPs in 5-day-old and 10-day-old chickens are summarized (n = 5). Each bar represents the mean of data ± S.D.
5-day-old chickens (Fig. 4A and C). At 10 days of age, the EJP was more prominent (Fig. 4B and C) but was still less than that observed in 12week-old chickens (see Fig. 2B and C). Pretreatment of tissue preparation obtained from 10-day-old chickens with a muscarinic cholinoceptor blocker, atropine (0.5 μM), completely abolished the EJP (Fig. 5). Atropine did not
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Fig. 6. Effects of suramin on EJP evoked by EFS. (A) Typical recordings of EFS-evoked responses in longitudinal smooth muscle of the anterior mesenteric artery of 10-dayold chickens in the absence (control) or presence of suramin (500 μM) are shown. Similar results were obtained in five independent experiments. (B) The effects of suramin (500 μM) on the EFS-evoked EJP are summarized (n = 5). Each bar represents the mean of data ± S.D.
significantly affect resting membrane potential (−33.3 ± 1.2 mV, n = 10). In contrast, a non-specific purinoceptor antagonist, suramin (500 μM), had no effect on the EFS-evoked EJP (Fig. 6). Molecular identification of purinoceptors in the chicken anterior mesenteric artery by RT-PCR We then examined expression of purinoceptor isoforms (nomeculture following Alexander et al. 2008) in the anterior mesenteric artery of 10-day-old chickens by using RT-PCR. Amplified products of P2X1, P2X4, P2X5, P2Y1, P2Y5 and P2Y6 were observed in appropriate sizes (Fig. 7A). Similar expression pattern of purinoceptor isoforms were observed in the anterior mesenteric artery of 12-week-old chickens (Fig. 7B). Discussion
Fig. 5. Effects of atropine on EJP evoked by EFS. (A) Typical recordings of EFS-evoked responses in longitudinal smooth muscle of the anterior mesenteric artery of 10-dayold chickens in the absence (control) or presence of atropine (0.5 μM) are shown. Similar results were obtained in five independent experiments. (B) The inhibitory effects of atropine (0.5 μM) on the EFS-evoked EJP are summarized (n = 5). Each bar represents the mean of data ± S.D. *p b 0.05, compared to the control.
We previously showed that perivascular nerve stimulation evoked EJP of longitudinal smooth muscles in anterior mesenteric arteries of adult chickens (Khalifa et al., 2005). In the present study, we investigated postnatal development of excitatory innervation to longitudinal smooth muscles. The major findings are (i) a welldeveloped longitudinal smooth muscle layer exists in the anterior mesenteric artery of 1-day-old chickens, (ii) perivascular nerve stimulation failed to evoke response of membrane potential in the smooth muscle of 1-day-old chickens, although it caused a large EJP in 12-week-old chickens, (iii) the smooth muscle of 1-day-old chickens showed depolarization in response to exogenously applied ACh or ATP, (iv) EFS-evoked EJP was observed in 5-day-old chickens and became more prominent at the age of 10 days, (v) the EJP in 10-dayold chickens was blocked by a muscarinic ACh receptor blocker (atropine), whereas blockade of purinoceptors by a non-specific purinoceptor antagonist, suramin, had no effect, and (vi) several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in the smooth muscle of adult chicken (Khalifa et al., 2005), were expressed in the anterior mesenteric artery of 10day-old chickens. These results demonstrate that excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric
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Fig. 7. Identification of purinoceptor isoforms in the chicken anterior mesenteric artery by RT-PCR. Homogenized samples from the anterior mesenteric artery of 10-day-old (A) and 12-week-old (B) chickens were used for RT-PCR. Amplified products of P2X1, P2X4, P2X5, P2Y1, P2Y5 and P2Y6 purinoceptors were observed in appropriate sizes in the artery.
artery is not established at birth but developed during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in the smooth muscle. In the rat portal vein, the longitudinal smooth muscle layer is not found at birth (Ts'Ao et al., 1971) but becomes apparent at 2 weeks of age (Lundberg et al., 1976). Taking into consideration that the longitudinal smooth muscle layer develops after birth in the vein, it was of interest to determine whether the chicken anterior mesenteric artery possesses a longitudinal smooth muscle layer at birth. Hematoxylin and eosin staining of cross-sections of the artery revealed that a well-developed longitudinal smooth muscle layer exists at 1 day of age. To our knowledge, this is the first demonstration of the presence of an outer longitudinal muscle layer in the artery of a newly hatched chicken. Perivascular nerve stimulation failed to produce any responses in the longitudinal smooth muscle of 1-day-old chickens. The same stimulus protocol substantially induced EJP in preparations isolated from 12-week-old chickens. It thus seems likely that the EFS applied to the preparations is sufficient to stimulate perivascular nerves. In accordance with this, multiple pulse stimulation at 20 Hz was still insufficient to evoke membrane response in 1-day-old chickens. One possible explanation for the failure of EFS to induce membrane response is a defect in responsiveness of the smooth muscle to excitatory neurotransmitters, including ACh and ATP. However, this is not the case, since exogenous application of ACh or ATP could induce depolarization even in preparations obtained from 1-day-old chickens (Fig. 3). Alternatively, it is reasonable to assume that excitatory neuromuscular transmission is not completed and/or effective innervations are not well-established in newly hatched chickens. This assumption is consistent with results of a study by Ljung and
Stage (1975), showing that responsiveness of vascular smooth muscle to exogenous noradrenaline is established before the development of sympathetic innervation. Although perivascular nerve stimulation failed to elicit EJP of the longitudinal smooth muscle in newly hatched chickens, it was capable of eliciting a slight EJP in 5-day-old chickens (Fig. 4A). As preparation obtained from 10-day-old chicken showed more prominent EJP in response to perivascular nerve stimulation (Fig. 4B), it can be considered that excitatory neuromuscular transmission gradually develops during the early postnatal period. It was previously demonstrated that both cholinergic and purinergic components are involved in depolarizing response of the longitudinal smooth muscle in mature chickens (Khalifa et al., 2005). However, a non-specific purinoceptor antagonist, suramin, had no effect on the depolarizing response of 10-day-old chickens (Fig. 6), although purinoceptors found in the anterior mesenteric artery of adult chickens were already expressed in the artery at the age (Fig. 7). In addition, the EJP was completely abolished by a muscarinic ACh receptor antagonist, atropine (Fig. 5), while the antagonist only partially attenuated EJP observed in mature chickens (Khalifa et al., 2005). These results suggest that cholinergic nerves exclusively innervate the longitudinal smooth muscle in the early postnatal period. Thus, it is concluded that each nerve component does not develop uniformly; cholinergic innervation, which begins as early as 5 days of age, precedes purinergic innervation. At present, the timing of purinergic innervation is unclear. In relation with this, however, our previous study showed that purinergic excitatory neuromuscular transmission is still not obvious in chicken aged 5 weeks (Alkayed et al., 2009). Recently, we have demonstrated that activation of perivascular purinergic component in relatively young chickens (5 weeks of age) causes prominent hyperpolarization, not depolarization, in longitudinal smooth muscle of the anterior mesenteric artery (Alkayed et al., 2009). The events associated with the hyperpolarization consist of ATP release from nerve terminals, its binding to endothelial P2X purinoceptors, generation of nitric oxide (NO) in endothelial cells, diffusion of NO to the longitudinal smooth muscle, and activation of guanylate cyclase. The present results also suggest that the inhibitory purinergic component is not established in newly hatched chickens. Together with our previous findings (Alkayed et al., 2009; Khalifa et al., 2005), our data provide an overview of developmental changes in neural components that control the longitudinal smooth muscle of the chicken anterior mesenteric artery as summarized in Table 1. The physiological significance of the age-dependent shift of purinergic regulation, i.e., shift from inhibitory to excitatory, is unclear at present. The longitudinal smooth muscle would play a minor role, if any, in regulation of vascular tone, since contraction of longitudinal smooth muscle had little effect on perfusion pressure in the isolated chicken mesenteric artery (Bell, 1969). In the case of the posterior mesenteric artery of chickens, the longitudinal muscle layer was found only in females (unpublished observation). Remarkably, occurrence of the longitudinal muscle layer in female chickens became obvious when chickens began egg production. Therefore, it is reasonable to assume that the longitudinal smooth muscle confers resistance to tension applied to blood vessels. Based on this assumption, the age-dependent shift of purinergic regulation can be explained as an adaptive change against putative higher tension
Table 1 Developmental changes in neuronal components of the chicken anterior mesenteric artery. Neuronal control
1 day
10 days
5 weeks
12 weeks
Excitatory cholinergic component Excitatory purinergic component Inhibitory purinergic component (Endothelium-dependent)
− − −
± − −
+ − +
+ + ±
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applied to the anterior mesenteric artery in mature chickens. Further study is needed to elucidate the physiological roles of the longitudinal smooth muscle and developmental changes in regulatory neural components. Conclusion The present study suggests that excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but developed during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in the smooth muscle. Conflict of interest statement The authors declare that there are no conflicts of interest.
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