Effect of central thyrotropin-releasing hormone on pancreatic blood flow in rats

Regulatory Peptides 121 (2004) 57 – 63 www.elsevier.com/locate/regpep

Effect of central thyrotropin-releasing hormone on pancreatic blood flow in rats Manabu Goto a, Masashi Yoneda b,*, Kimihide Nakamura a, Akira Terano b, Masakazu Haneda a a

b

Second Department of Medicine, Asahikawa Medical College, Asahikawa, Japan Department of Gastroenterology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan Received 30 September 2003; received in revised form 7 April 2004; accepted 21 April 2004 Available online 1 June 2004

Abstract Central neuropeptides play a role in physiological regulation through the autonomic nervous system. Thyrotropin-releasing hormone (TRH) is a neuropeptide distributed throughout the central nervous system and acts as a neurotransmitter to regulate gastric and hepatic functions through vagal-cholinergic pathways. In this study, the central effect of TRH on pancreatic blood flow was investigated in urethaneanesthetized rats. Pancreatic blood flow was determined by laser Doppler flowmetery. After measurement of basal blood flow, a stable TRH analog, RX 77368 (1 – 50 ng) or saline was injected intracisternally. Pancreatic blood flow was observed for 120 min thereafter. In some experiments, pretreatnent with atropine methyl nitrate (0.15 mg/kg, i.p.), N G-nitro-L-arginine-methyl ester (10 mg/kg, i.v.), or 6hydroxydopamine (6-OHDA;180 mg/kg, i.p.), or subdiaphragmatic vagotomy was performed. Intracisternal injection of TRH analog dosedependently increased pancreatic blood flow with a peak response occurring 30 min after injection. The stimulatory effect of TRH analog on pancreatic blood flow was blocked by vagotomy, atropine, and NG-nitro-L-arginine-methyl ester, but not by 6-hydroxydopamine. Intravenous administration of the TRH analog did not influence pancreatic blood flow in the same animal model. These results indicate that TRH acts in the central nervous system to stimulate pancreatic blood flow through vagal-cholinergic and nitric oxide-dependent pathways. D 2004 Elsevier B.V. All rights reserved. Keywords: Neuropeptide; Autonomic nervous system; Central nervous system (CNS); Thyrotropin-releasing hormone (TRH); Nitric oxide

1. Introduction Convergent neuroanatomical, electrophysiological, and neuropharmacological evidence has suggested a role for the central and autonomic nervous systems in the regulation of pancreatic functions [1 – 3]. Neuropeptides have recently been recognized as neurotransmitters in the central and peripheral nervous systems [4 – 8], and centrally acting neuropeptides have been reported to regulate a variety of physiological functions [9– 11]. In particular, the effect of central thyrotropin-releasing hormone (TRH) on physiological, pharmacological, and pathophysiological regulation of gastrointestinal tract functions has been reported [12]. With respect to the stomach, central administration of TRH and a TRH analog stimulate gastric motility and secretions Abbreviations: TRH, thyrotropin-releasing hormone; 6-OHDA, 6hydroxydopamine; L-NAME, N G-nitro-L-arginine-methyl ester. * Corresponding author. Tel.: +81-282-86-2724; fax: +81-282-867761. E-mail address: [email protected] (M. Yoneda). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.04.006

[13,14]. Gastric mucosal blood flow is also stimulated by intracerebroventricular and intracisternal injection of TRH and a TRH analog through vagal-cholinergic pathways [15,16]. In the brain, TRH-immunoreactive nerve fibers and terminals, and TRH receptors have been localized in the hypothalamus and the dorsal vagal complex, which includes the vagal motor nucleus and the nucleus of the solitary tract [17,18]. These nuclei are important sites for autonomic nervous regulation of the digestive system including the pancreas [19 – 21]. TRH causes a direct postsynaptic excitatory effect on preganglionic neurons in the dorsal motor nucleus and increases efferent vagal activity [22,23]. Moreover, the pancreas is densely innervated [21,24 – 26], and the autonomic nervous system influences pancreatic microcirculation in animal models [27 –29]. We have recently shown the role of central TRH in the regulation of hepatic functions. Intracisternal injection of a TRH analog stimulates hepatic proliferation and blood flow, and elicits a protective effect against experimental liver damage through vagalcholinergic pathways [30 – 33].

58

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63

In regard to the pancreas, central injection of TRH stimulates pancreatic exocrine and endocrine secretions [34,35]. These findings led us to speculate that TRH acts in the central nervous system to modify pancreatic blood flow through vagal-cholinergic pathways. The present study addressed this question by examining the effect of intracisternal injection of TRH on pancreatic blood flow in rats.

2. Materials and methods 2.1. Ethics All protocols of the present study were approved by the Animal Care Committee of Asahikawa Medical College, and were in accordance with the National Institute of Health of Japan ‘Guide for the Care and Use of Laboratory Animals’. 2.2. Animals Male Wistar rats weighing 240– 275 g (SLC, Shizuoka, Japan) were housed in group cages under condition of controlled temperature (22 –24 jC) and illumination (12h light cycle starting at 6 AM) for at least 7 days before experiments. Animals were maintained on standard laboratory chow (Oriental Yeast, Tokyo, Japan) and tap water ad libitum. Experiments were performed in rats deprived of food for 24 h but given free access to water until commencement of the study. 2.3. Chemicals The following substances were used: the stable TRH analog, RX 77368, p-Glu-His-(3,3V-dimethyl)-Pro-NH2 (Reckitt & Colman, Kingdom-upon-Hill, England), atropine methyl nitrate (Sigma, St Louis, MO), NG-nitro-L-argininemethyl ester (L-NAME; Sigma), 6-hydroxydopamine (6OHDA; Aldrich, Milwaukee, WI). RX 77368 has similar binding characteristics as the authentic TRH on rat brain membranes, but is more stable compared with the natural TRH [36,37]. RX 77368 was aliquoted in 0.5% bovine serum albumin (Sigma) and 0.9% saline at the concentration of 1.5 nmol/Al, and kept frozen at 20 jC. The stock solution was diluted in 0.9% saline (pH 7.4) before the experiment. 2.4. Experimental design Rats were anesthetized with urethane (1.5 g/kg, i.p.) and underwent tracheotomy, and then PE-260 tubing (Clay Adams, B.D., Parsippany, NJ) was inserted into the trachea to ensure a patent airway. The femoral artery was cannulated with PE-50 tubing (Clay Adams, B.D.) to enable continuous monitoring and recording of arterial blood pressure using a pressure transducer (Uniflow, Baxter, Valencia, CA), a

pressure amplifier (PA-001, Star Medical, Tokyo, Japan), and a computer (Macintosh G4, Apple Computer, Cupertino, CA) equipped with a data recording and analysis system (MacLab, AD Instruments, Castle Hill, Australia). Rats were mounted on ear bars of a stereotaxic apparatus (Kopf model 900, David Kopf Instruments, Tujunga, CA), and each rat was positioned to expose the abdomen. After making a 3-cm midline abdominal incision, the pylorus was ligated and a cannula was placed into the nonglandular portion of the stomach to divert gastric secretion, so as to avoid the possibility of inducing duodenal acidification and gastric distention which may influence pancreatic functions. Then, a probe (diameter 6 mm, type H, Advance, Tokyo Japan) of a laser Doppler flowmeter (ALF 21, Advance) was placed on the surface of pancreatic body. The flow signal was averaged with a 3-s time constant and recorded using a computer (Macintosh G4) equipped with a data recording and analysis system (MacLab). Pancreatic blood flow was expressed relative to the basal. Body temperature was kept at 37 jC by external heating and the pancreatic surface was continuously rinsed with 0.9% saline (37 jC, pH 7.4) to keep moist. After completing the surgical preparation, the animals were left undisturbed for 60 min to stabilize temperature, heart rate, and arterial blood pressure. After measurement of basal pancreatic blood flow for 20 min, either the TRH analog, RX 77368 (1, 3, 5, 10, 30, or 50 ng), or 0.9% saline vehicle was injected intracisternally in a 10-Al volume using a 50-Al Hamilton microsyringe (Hamilton, Reno, NV). The doses of TRH analog were selected based on previous studies that indicated the central effects of RX 77368 on gastric and hepatic functions [9,13,14,30– 32]. The intracisternal point of injection was found by palpation a few millimeters behind the caudal end of the skull. The accuracy of intracisternal injection was ascertained by aspiration of cerebrospinal fluid before and after the injection. A change in pancreatic blood flow was observed for 120-min postinjection. In another experiment, changes in pancreatic blood flow were measured after injection of RX 77368 (30 ng) or 0.9% saline vehicle in the femoral vein. 2.5. Effect of atropine, 6-OHDA, L-NAME and subdiaphragmatic vagotomy on TRH analog-induced modulation of pancreatic blood flow Atropine methyl nitrate (0.15 mg/kg) dissolved in 0.9% saline was injected intraperitoneally 15 min before RX77368 injection in a 1.0-ml/kg volume. 6-OHDA, which is known to chemically denervate the sympathetic nerves, dissolved in 0.9% saline was intraperitoneally injected twice (100 mg/kg on the first day, 80 mg/kg on the fourth day), and intracisternal injection of TRH analog was performed on the seventh day [38]. L-NAME (10 mg/kg) dissolved in 0.9% saline was injected intravenously 15 min before RX77368 injection in a 1.0-ml/kg volume. To evaluate the specificity of the effect of L-NAME on pancreatic blood

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63

59

flow modified by central TRH analog, L-arginine (Sigma, 800 mg/kg bolus i.v. followed by 200 mg/kg/h continuous i.v. throughout the experiment) was administered just before the administration of L-NAME in an additional group. Either subdiaphragmatic vagotomy or sham operation was performed under light ether anesthesia 120 min before RX77368 injection. 2.6. Statistical analysis Results are expressed as mean F S.E. Comparison to basal of pancreatic blood flow and arterial blood pressure after RX77368 injection was calculated by analysis of variance (ANOVA)-repeated measurement followed by Fisher’s protected least significant difference test. Comparison between two independent groups was calculated by Mann –Whitney U-test. Multiple group comparisons were performed by ANOVA followed by Fisher’s protected least significant difference test. A P value < 0.05 was considered statistically significant.

Fig. 2. Dose – response of intracisternal TRH analog effect on pancreatic blood flow in urethane-anesthetized rats. After a measurement of basal pancreatic blood flow by laser Doppler flowmetry, either saline or RX 77368 (1 – 50 ng) was injected intracisternally. For other details, see Fig. 1 legend. Each bar represents mean F S.E. of % change of pancreatic blood flow at 30 min after injection. *P < 0.05, **P < 0.01 compared with salineinjected group.

3. Results 3.1. Effect of intracisternal injection of the TRH analog on pancreatic blood flow Compared to saline, intracisternal injection of 30 ng RX 77368 increased pancreatic blood flow, with peak response of 45 F 6% over basal by 30 min after injection. Pancreatic blood flow returned to basal by 90 min after injection (Fig. 1). The stimulatory action of RX 77368 on pancreatic blood flow was dose-dependent 30 min after injection (% change from basal line: vehicle 1 F 2; RX 77368 1 ng: 5 F 7; 3 ng: 15 F 6; 5 ng: 32 F 9; 10 ng: 40 F 9; 30 ng: 45 F 6; 50 ng: 42 F 8) (Fig. 2). RX 77368 (30 ng) injected intravenously had no effect on pancreatic blood flow (Table 1).

3.2. Effect of atropine, 6-OHDA, L-NAME, and subdiaphragmatic vagotomy on pancreatic blood flow in response to central TRH analog Subdiaphragmatic vagotomy performed 120 min before, atropine methyl nitrate (0.15 mg/kg) injected intraperitoneally 15 min before, or L-NAME (10 mg/kg) injected intravenously 15 min before intracisternal injection of RX77368 prevented the increased in pancreatic blood flow in response to TRH analog (30 ng) (Fig. 3A –C). 6-OHDA intraperitoneal injection (100 mg/kg 7 days before and 80 mg/kg 4 days before) did not modify the stimulation of pancreatic blood flow induced by intracisternal RX 77368 (30 ng) (Fig. 3D). L-arginine (800 mg/kg bolus i.v. followed by 200 mg/kg/h continuous i.v. infusion) abolished the inhibitory effect of L-NAME on central TRH analog-induced stimulation of pancreatic blood flow (Fig. 3D). 3.3. Effect of intracisternal TRH analog on mean arterial blood pressure

Fig. 1. Effect of intracisternal (i.c.) thyrotropin-releasing hormone (TRH) analog, RX 77368 (30 ng) on pancreatic blood flow in urethaneanesthetized rats. After measurement of basal pancreatic blood flow by laser Doppler flowmetry, either saline or RX 77368 (30 ng) was injected intracisternally. Pancreatic blood flow was monitored thereafter for 120 min. Each point represents mean F S.E. *P < 0.05, **P < 0.01 compared with respective basal period.

Mean arterical blood pressure was increased slightly by 15 min after intracisternal injection of RX 77368 (30 ng), and then returned to basal by 60 min (Table 2). Although pretreatment with atropine (15 min), L-NAME (15 min), or subdiaphragmatic vagotomy (120 min) did not influence intracisternal TRH analog-induced elevation of mean arterial blood pressure, 6-OHDA pretreatment (100 mg/kg 7 days before and 80 mg/kg 4 days before) inhibited the rise in mean arterial blood pressure induced by central injection of the TRH analog (Table 2). Although 6-OHDA pretreatment suppressed and L-NAME treatment enhanced mean arterial blood pressure, respectively, subdiaphragmatic va-

60

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63

Table 1 Effect of intravenous injection of TRH analog, RX 77368, on pancreatic blood flow in urethane-anesthetized rats Time after intravenous injection (min) 0

15

30

45

60

75

90

105

120

98 F 4 100 F 3

103 F 4 101 F 7

100 F 6 103 F 4

96 F 4 98 F 4

102 F 4 103 F 6

103 F 5 96 F 4

Change of pancreatic blood flow (%) Saline RX 77368

100 F 0 100 F 0

98 F 3 101 F 4

101 F 2 98 F 4

Values are mean F S.E. of five rats per group. After a basal observation, saline or RX 77368 (30 ng) was injected intravenously. A change of pancreatic blood flow was monitored thereafter for 120 min.

gotomy and atropine treatment did not modify basal mean arterial blood pressure (Table 2). Intravenous administration of the TRH analog (30 ng) did not alter mean arterial blood pressure (data not shown).

4. Discussion In the present study, we demonstrated that intracisternal injection of 3– 50 ng of the stable TRH analog, RX 77368 stimulated pancreatic blood flow in urethane-anesthetized rats as determined by laser Doppler flowmetry, a device capable of detecting a relative change of regional tissue blood flow. The stimulatory effect of intracisternal injection of the TRH analog was dose-dependent over a range of 3 – 30 ng. Intracisternal RX 77368 (30 ng) injection significantly increases pancreatic blood flow with a peak response in the first 30-min observation period, returning to basal by 90 min. The 30-ng dose induced a maximal response, with a percent

change of 45 F 6%; injection of a 50-ng dose elicited no further enhancement of pancreatic blood flow. In contrast, RX 77368 injected intravenously at the maximal effective dose given intracisternally had no effect on pancreatic blood flow. These results indicate that RX 77368, injected into the cisternal magna, acts in the central nervous system to stimulate pancreatic blood flow. Further, this central effect is not influenced by leakage of the stimulatory dose into the peripheral circulation. The pathways through which central administration of the TRH analog enhanced pancreatic blood flow were investigated in the present study. The stimulatory effect was abolished by subdiaphragmatic vagotomy and atropine treatment, whereas 6-OHDA pretreatment had no effect. These results indicate that the central action of TRH is mediated through vagal-cholinergic pathways. Similarly, previous reports have indicated that central TRH-induced stimulation of gastric and hepatic functions is mediated through vagalcholinergic dependent mechanisms [9,13,14,30 – 33]. More-

Fig. 3. Effect of subdiaphragmatic vagotomy (A), atropine (B), N G-nitro-L-arginine-methyl ester (L-NAME; C), and 6-hydroxydopamine (6-OHDA; D) on intracisternal TRH analog (30 ng)-induced enhancement of pancreatic blood flow in urethane-anesthetized rats. Subdiaphragmatic vagotomy was performed 120 min before, atropine methyl nitrate (0.15 mg/kg, i.p.) and L-NAME (10 mg/kg, i.v.) were injected 15 min before, and 6-OHDA was injected 7 days before (100 mg/kg) and 4 days before (80 mg/kg) the intracisternal injection of RX 77368 (30 ng). L-arginine (800 mg/kg bolus i.v. followed by 200 mg/kg/ h continuous i.v. infusion) was administered just before L-NAME injection. For details see legend of Fig. 1. Each point represents the mean F S.E. *P < 0.05, **P < 0.01 compared with respective basal period.

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63

61

Table 2 Effect of intracisternal TRH analog, RX 77368, on mean arterial pressure in urethane-anesthetized rats Time after intracisternal injection (min) 0

15

30

45

60

75

90

105

120

Mean arterial pressure (mm Hg) No treatment Saline RX 77368

101 F 6 98 F 5

99 F 7 108 F 4

102 F 5 116 F 5*

100 F 6 108 F 4*

104 F 5 100 F 4

98 F 7 96 F 5

105 F 6 101 F 6

96 F 8 98 F 7

103 F 5 100 F 6

Vagotomy Saline RX 77368

103 F 6 99 F 5

98 F 3 107 F 6

99 F 4 118 F 7*

100 F 5 109 F 6*

101 F 4 102 F 6

101 F 7 99 F 5

99 F 7 97 F 7

96 F 8 101 F 6

101 F 7 98 F 5

Atropine Saline RX 77368

98 F 5 101 F 4

102 F 4 109 F 7

101 F 6 120 F 6*

99 F 6 107 F 6*

102 F 5 101 F 5

99 F 8 102 F 4

103 F 5 100 F 4

99 F 5 103 F 4

100 F 5 102 F 4

L-NAME Saline RX 77368

110 F 6 109 F 7

109 F 3 114 F 4

111 F 6 126 F 6*

110 F 5 118 F 5*

108 F 5 110 F 5

106 F 7 108 F 7

108 F 6 111 F 6

112 F 8 108 F 5

109 F 7 109 F 5

6-OHDA Saline RX 77368

72 F 4 69 F 4

71 F 6 70 F 5

69 F 5 71 F 5

75 F 4 74 F 5

70 F 4 69 F 5

72 F 4 72 F 4

69 F 4 71 F 6

68 F 5 70 F 4

70 F 5 68 F 5

Values are means F S.E. of five rats per group. After basal observation, saline vehicle or RX 77368 (30 ng) was injected intracisternally. Systemic blood pressure was monitored thereafter for 120 min. Subdiaphragmatic vagotomy was performed 120 min before, atropine methyl nitrate (0.15 mg/kg, i.p.) and L-NAME (10 mg/kg, i.v.) were injected 15 min before, and 6-OHDA was injected 7 days before (100 mg/kg) and 4 days before (80 mg/kg) the intracisternal injection of RX 77368 (30 ng). * P < 0.05 compared with respective basal period.

over, the stimulatory effect of TRH injected centrally on pancreatic endocrine and exocrine secretion was blocked by vagotomy [34,35], and electrical vagal stimulation induces pancreatic vasodilatation of resistance vessels [29]. Taken together, these findings suggest that central TRH activates parasympathetic outflow to the digestive system. This is further supported by electrophysiological studies that show central TRH-induced activation of vagal efferent discharge in rats [22]. In regard to the stomach, central administration of TRH increases nitric oxide release through activation of the vagal nerve [39], and increases gastric mucosal blood flow via release of nitric oxide [16,40]. Further, stimulation of hepatic blood flow by central administration of the TRH analog also depends on nitric oxide synthesis [34]. In the present study, the role of nitric oxide in TRH analoginduced increase in pancreatic blood flow was investigated by blocking nitric oxide synthesis using L-NAME. L-NAME reversed the stimulatory effect of central TRH analog on pancreatic blood flow, suggesting involvement of nitric oxide in central TRH-induced modulation of pancreatic blood flow. In agreement with previous findings [15,16], intracisternal injection of TRH analog also stimulated mean systemic arterial blood pressure. The increase in systemic arterial blood pressure, but not pancreatic blood flow, was attenuated within 60 min of intracisternal injection of TRH analog. Moreover, central TRH-induced stimulation of systemic blood pressure, but not pancreatic blood flow, was blocked

by 6-OHDA pretreatment. In addition, although central TRH-stimulated pancreatic blood flow was abolished by vagotomy, atropine and L-NAME, none of these treatments had any effect on TRH-induced elavation of blood pressure. Taken together, it is not likely that central TRH-induced increase of pancreatic blood flow is mediated by modulation of the peripheral circulation. Although a previous study showed that intravenous administration of TRH elevated mean arterial blood pressure [41], intravenous injection of the TRH analog had no effect in the present study. The likely reason for this is that we injected a substantially smaller dose (30 ng) than did the earlier study (2 mg/kg). Further, in the earlier study [41], a relatively large dose of TRH may elicit hormonal changes that themselves may result in an elevation of mean arterial pressure. Centrally injected TRH induces parasympathetic nerve activation [22] by acting in the medullary nuclei including the dorsal vagal complex and nucleus ambiguous [42,43], which are important sites for parasympathetic outflow [44]. Although microinjections of TRH analog into specific brain nuclei were not performed in the present study, the dorsal vagal complex, including the vagal motor nucleus and the nucleus of the solitary tract, are likely sites of the TRHinduced stimulation of pancreatic blood flow. TRH-immunoreactive nerve terminals and receptors are localized in the dorsal vagal complex [18,45], and various central TRHinduced gastric functions are reproduced by microinjection into the dorsal vagal complex [42,43]. Furthermore, we recently found that microinjection of TRH analog into the

62

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63

dorsal vagal complex increases hepatic blood flow [46], while another group showed that TRH injection stimulated pancreatic exocrine secretion [35]. In addition, vagotomy blocked the stimulatory effect of central TRH analog on pancreatic blood flow. Recent studies using radioisotopes have demonstrated that within 5-min peptides injected intracisternally ultimately reach the medullary nuclei, including the dorsal vagal complex [47]. These facts support that the dorsal vagal complex may be the specific site for TRH action on pancreatic blood flow and our preliminary data supports this hypothesis [48]. Although blood levels of thyrotropin-stimulating hormone were not measured in the present study, endocrine mechanisms are unlikely to be involved in central TRH analog-induced modulation of pancreatic blood flow, because intravenous TRH had no effect. Instead, subdiaphragmatic vagotomy completely abolished the increase in pancreatic blood flow induced by central TRH analog. TRH has previously been shown to elicit various CNSmediated actions unrelated to its neuroendocrine-mediated effects, including modification of behavior, thermaregulation, systemic blood pressure, food intake, and cardiac, respiratory and gastric functions. These central actions of TRH are mediated through the autonomic nervous system [9,12,34,49 – 51]. In summary, the data of our present study indicate that central TRH analog acts solely in the brain to induce potent stimulation of pancreatic blood flow. TRH action is mediated through vagal-cholinergic, nitric oxide-dependent pathway. These findings support a physiological role of central TRH in the regulation of pancreatic microcirculation. Although the therapeutic efficacy of increasing pancreatic blood flow in conditions of pancreatic damage is controversial [52,53], our study reveals a technique by which to investigate the effect in an animal model, such as ceruleininduced pancreatitis rat model of acute pancreatitis.

[4]

[5]

[6] [7] [8]

[9]

[10]

[11]

[12] [13]

[14]

[15]

[16] [17]

[18]

[19]

Acknowledgements The authors thank Prof. K.C. Kent Lloyd, DVM, PhD (University of California Davis) for editorial assistance. Grant support: Supported in part by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 15590681) and Japan Smoking Research Foundation.

References [1] Miller RE. Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the Islets of Langerhans. Endocr Rev 1981; 2:471 – 94. [2] Nishi S, Seino Y, Ishida H, et al. Vagal regulation of insulin, glucagon and somatostatin secretion in vitro in the rat. J Clin Invest 1987;89: 1191 – 6. [3] Kurose T, Seino Y, Nishi S, et al. Mechanism of sympathetic neural

[20]

[21]

[22]

[23]

[24]

[25]

regulation of insulin, somatostatin, and glucagon secretion. Am J Physiol 1990;258:E220 – 7. Smith JR, La HT, Chesnut RM, et al. Thyrotropin-releasing hormone: stimulation of colonic activity following intracerebroventricular administration. Science 1977;196:660 – 2. Brown M, Tache´ Y, Fisher D. Central nervous system action of bombesin: mechanism to induce hyperglycemia. Endocrinology 1979;105: 660 – 5. Brown M, Rivier J, Vale W. Somatostatin: central nervous system actions on glucoregulation. Endocrinology 1979;104:1709 – 15. Tache´ Y. CNS peptides and regulation of gastric acid secretion. Annu Rev Physiol 1988;50:19 – 39. Tache´ Y, Goto Y, Gunion MW, et al. Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor. Science 1983;222:935 – 7. Tache´ Y, Yang H. Brain regulation of gastric acid secretion by peptides. Sites and mechanisms of action. Ann N Y Acad Sci 1990; 597:128 – 45. Lenz HJ, Burlage M, Raedler A, et al. Central nervous system effects of corticotropin-releasing factor on gastrointestinal transit in the rat. Gastroenterology 1988;94:598 – 602. Lenz HJ, Druge G. Neurohumoral pathways mediating stress-induced inhibition of gastric acid secretion in rats. Gastroenterology 1990;98: 1490 – 2. Tache´ Y, Vale W, Brown M. Thyrotropin-releasing hormone-CNS action to stimulate gastric acid secretion. Nature 1980;287:149 – 51. Garrick T, Buack S, Veiseh A, Tache´ Y. Thyrotropin-releasing hormone (TRH) acts centrally to stimulate gastric contractility in rats. Life Sci 1987;40:649 – 57. Yoneda M, Tache´ Y. Central thyrotropin-releasing factor analog prevents ethanol-induced gastric damage through prostaglandins in rats. Gastroenterology 1992;102:1568 – 74. Thiefin G, Tache´ Y, Leung FW, et al. Central nervous system action of thyrotropin-releasing hormone to increase gastric mucosal blood flow in the rat. Gastroenterology 1989;97:405 – 11. Tanaka T, Guth P, Tache´ Y. Role of nitric oxide in gastric hyperemia induced by central vagal stimulation. Am J Physiol 1993;264:G280 – 4. Palkovits M, Mezey E, Eskay RL, et al. Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropinreleasing hormone-containing raphe neurons. Brain Res 1986; 373:246 – 51. Rinaman L, Miselis RR. Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on the dentrites of gastric vagal motoneurons in the rat. J Comp Neurol 1990;294:235 – 51. Gillis RA, Quest JA, Pagani FD, et al. Central centers in the central nervous system for regulating gastrointestinal motility. In: Wood JD, editor. Handbook of physiology. Bethesda: American Physiological Society; 1989. p. 621 – 83. Rogers RC, Hermann GE. Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study. J Auton Nerv Syst 1983;7:165 – 74. Streefland C, Meas FW, Bohus B. Autonomic brainstem projections to pancreas: a retrograde transneural viral tracing study in the rat. J Auton Nerv Syst 1998;74:71 – 81. O-Lee TJ, Wei JY, Tache´ Y. Intracisternal TRH and RX 77368 potently activate gastric vagal efferent discharge in rats. Peptides 1997; 18:213 – 9. Travagli RA, Gillis RA, Vicini S. Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro. Am J Physiol 1992;263:G508 – 17. Luiten PG, Ter Horst G, Buijs RM, et al. Autonomic innervation of the pancreas in diabetic and non-diabetic rats. A new view on intramural sympathetic structural organization. J Auton Nerv Syst 1986; 15:33 – 44. Ushiki T, Ide C. Autonomic nerve networks in the rat exocrine pancreas as revealed by scanning and transmission electron microscopy. Arch Histol Cytol 1988;51:71 – 81.

M. Goto et al. / Regulatory Peptides 121 (2004) 57–63 [26] Ushiki T, Watanabe S. Distribution and ultrastructure of the autonomic nerves in the mouse pancreas. Microsc Res Tech 1997;37:399 – 406. [27] Hickson JC. The secretory and vascular response to nervous and hormonal stimulation in the pancreas of the pig. J Physiol 1970; 206:299 – 322. [28] Ahren B, Veith RC, Paquette TL, et al. Sympathetic nerve stimulation versus pancreatic norepinephrine infusion in the dog: 2. Effects on basal release of somatostatin and pancreatic polypeptide. Endocrinology 1987;121:332 – 9. [29] Holst JJ, Rasmussen TN, Schmidt P. Role of nitric oxide in neurally induced pancreatic exocrine secretion in pigs. Am J Physiol 1994; 266:G206 – 13. [30] Yoneda M, Tamori K, Sato Y, et al. Central thyrotropin-releasing hormone stimulates hepatic DNA synthesis in rats. Hepatology 1997; 26:1203 – 8. [31] Tamori K, Yoneda M, Nakamura K, et al. Effect of intracisternal thyrotropin-releasing hormone on hepatic blood flow in rats. Am J Physiol 1998;274:G277 – 82. [32] Sato Y, Yoneda M, Nakamura K, et al. Protective effect of central thyrotropin-releasing hormone on carbon tetrachloride-induced acute hepatocellular necrosis in rats. J Hepatol 2003;39:47 – 54. [33] Yoneda M, Watanobe H, Terano A. Central regulation of hepatic function by neuropeptides. J Gastroenterol 2001;36:361 – 7. [34] Brown M. Thyrotropin releasing factor: a putative CNS regulator of the autonomic nervous system. Life Sci 1981;28:1789 – 95. [35] Okumura T, Taylor IL, Pappas TN. Microinjection of TRH analogue into the dorsal vagal complex stimulates pancreatic secretion in rats. Am J Physiol 1995;269:G328 – 34. [36] Hawkins EF, Engel WK. Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implication. Life Sci 1985;36:601 – 11. [37] Griffiths EC, McDermott JR, Smith AI. Mechanisms of brain inactivation of centrally-acting thyrotrophin-releasing hormone (TRH) analogues: a high-performance liquid chromatography study. Regul Pept 1982;5:1 – 11. [38] Thoenen H, Tranzer JP. Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Arch Exp Pathol Pharmacol 1968;261:271 – 88. [39] Saperas E, Mourelle M, Santos J, et al. Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats. Am J Physiol 1995;268:G895 – 9.

63

[40] Kiraly A, Suto G, Guth PH, Tache´ Y. Peripheral mediators involved in gastric hyperemia to vagal activation by central TRH analog in rats. Am J Physiol 1998;274:G170 – 7. [41] Alhan E, Kucuktulu U, Ercin C, et al. The effects of calcium channel blocker and thyrotropin releasing hormone on acute necrotizing pancreatitis in rats. Res Exp Med 1999;199:51 – 8. [42] Ishikawa T, Yang H, Tache´ Y. Medullary sites of action of the TRH analogue, RX 77368, for stimulation of gastric acid secretion in the rat. Gastroenterology 1988;95:1470 – 6. [43] Stephens RL, Ishikawa T, Weiner H, et al. TRH analogue, RX 77368, injected into dorsal vagal complex stimulates gastric secretion in rats. Am J Physiol 1988;254:G639 – 43. [44] Leslie RA, Gwyn DG, Hopkins DA. The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res Bull 1982;8:37 – 43. [45] Manaker S, Rizio G. Autoradiographic localization of thyrotropinreleasing hormone and substance P receptors in the rat dorsal vagal complex. J Comp Neurol 1989;290:516 – 26. [46] Yoneda M, Hashimoto T, Nakamura K, et al. Thyrotropin-releasing hormone in the dorsal vagal complex stimulates hepatic blood flow in rats. Hepatology 2003;38:1500 – 7. [47] Proescholdt MG, Hutto B, Brady LS, et al. Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat. Neuroscience 2000;95:577 – 92. [48] Yoneda M, Tamori K, Nakade Y, et al. Thyrotropin-releasing hormone TRH in the dorsal vagal complex (DVC) increases the pancreatic blood flow in rats (abst). Gastroenterology 2000;118(4):A82. [49] Feuerstein G, Hassen AH, Faden AI. TRH: cardiovascular and sympathetic modulation in brain nuclei of the rat. Peptides 1983;4:617 – 20. [50] Wei E, Sigel S, Loh H, Way EL. Thyrotrophin-releasing hormone and shaking behaviour in rat. Nature 1975;253:739 – 40. [51] Vijayan E, McCann SM. Suppression of feeding and drinking activity in rats following intraventricular injection of thyrotropin releasing hormone (TRH). Endocrinology 1977;100:1727 – 30. [52] Andrzejewska A, Dlugosz JW. The endothelin-1 receptor antagonists ameliorate histology and ultrastructural alterations in the pancreas and decrease trypsinogen activation in severe taurocholate pancreatitis in rats. Int J Exp Pathol 2003;84:221 – 9. [53] Kogire M, Inoue K, Higashide S, et al. Protective effects of endothelin-1 on acute pancreatitis in rats. Dig Dis Sci 1995;40:1207 – 12.