Neural monitoring system for circulating somatostatin in the hepatoportal area

REVIEW

ARTICLE

Nutrition Vol. 13, No. 3, 1997

Neural Monitoring System for Circulating Somatostatin in The Hepatoportal Area HAJIME

NAKABAYASHI,

MD, D MED SC1

From the Department of Internal Medicine, Graduate School of Medicine, Kanazawa University, Ishikawa, Japan Date accepted:

12 June 1996

ABSTRACT

If a peptide hormone secreted from the gastroenteropancreatic (GEP) system is monitored by the hepatic vagal nerve, the nerve can signal the central nervous system and thereby exert control on its target organs. In this review, we offer a line of evidence for the hypothesis. When a physiological dose of somatostatin (SS), one of the GEP hormones, was injected into the rat portal vein, the spike discharge rate in the hepatic afferent vagus increased significantly. This SSinduced activation of the vagus was completely abolished by a prior administration of our monoclonal antibody to SS receptor into the portal vein. We further disclosed a morphological basis for this neural reception to SS in the hepatoportal area: the neural bodies. located beneath the endothelium of the rat portal vein, preferentially bound the exogenous SS injected intraportally as revealed immunohistologically. The bodies contained a structure of the nerve fiber arbotizations resembling those of the afferent apparatus of Krause, on which the presence of SS receptor was confirmed histochemically using the anti-SS receptor antibody. These results provide a new insight into the receptor-mediated neural reception to GEP hormones in the hepatoportal area, implying the potential role of the reception in the GEP physiology. Nutrition 1997: 13:225-229. OElsevier Science Inc. 1997 Key words: somatostatin,

hepatoportal gastroentero

pancreatic, neural system

INTRODUCTlON

Somatostatin (SS), one of the brain-gut hormones, is a tetradecaPeptide distributed widely throughout the central and peripheral nervous systems as well as in the pancreas and gastrointestinal tract.’ The splanchnic organs are known to be the major source of circulating SS.’ Moreover, it has been reported that the processing of prosomatostatin is different in the tissues of the gastroenteropancreatic (GEP) system; SS is synthesized predominantly in the pancreas, stomach, duodenum, colon, and some enteric neurons, whereas SS-28 moiety that contains 14 amino acids extension at N-terminal of SS, represents more than 50% of total SS-like immunoreactivity (SLI) in the small intestine.* In fact, circulating forms of both SS and SS-28 have been identified in the portal vein.‘,* However, the role of circulating SS in physiology and in the pathophysiology of various disease states is not fully understood, although its role in nutrient homeostasis has been stressed? In the present review, our studies, which indicate that SS is sensed by receptors on neural elements associated with the

intrahepatic portal vein, are described. The studies also provide new insight into the means by which GEP hormones signal the central nervous system and thereby exert control on their target organs. THE AF’FERENT NERVOUS

SYSTEM

1N THE

HEPATOPORTALAREA

The hepatic afferent vagus is known to be receptive to changes of metabolite concentration (e.g., glucose), osmolality, and temperature in the portal vein blood as validated by behavioral and electrophysiological studies.5-7 (See also a review by Sawchenko and Friedman*.) The vagal afferent nerve fibers are distributed to the hepatoportal area.8 Studies with transmission electron microscopy and laser scanning confocal microscopy have recently demonstrated the terminal location of the fibers in the portal adventitia.’ Though the physiological implication of the terminal location of the hepatic vagal afferent nerve in the hepatoportal

Correspondence to: Hajime Nakabayashi, MD, D Med Sci, Department of Internal Medicine (II), School of Medicine, Kanazawa University, 131 Takara-machi, Kanazawa, Ishikawa 920, Japan.

Nutrition 13:225--229, 1997 OElsevier Science Inc. 1997 Printed in the USA. All rights reserved.

ELSEVIER

0899-9007/97/$17.00 PI1 SOS99-9007(96)00438-S

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injection. The SS-induced increases usually lasted for more than 120 min after the injection (Figs. 1 and 2),” confirming our previous results.‘” As shown in Figure 2, the discharge rate of the afferent spikes rose significantly from 56 2 3 impulses/ 5 s before the injection to 68 2 22, 118 j, 11, and 191 2 37 at 30, 60, and 90 min, respectively, after the injection.” The intraportal injection of the vehicle did not significantly change the discharge rate. Plasma SS levels in the portal vein at the time of the termination of the SS injection are assumed to reach about 200-230 pmol/L by calculation. The levels meet plasma SLI levels in the portal vein seen in various physiological situations such as meal ingestion (200-600 pmol/L) in rats and dogs. Moreover, the intraportal injection of 0.6 pmol SS also significantly facilitated the afferent discharge rate (data not shown). Thus, the results indicate that a physiological dose of SS delivered to the liver through the portal vein stimulates the afferent activity in the hepatic vagus, showing the vagal chemoreception that converts efficiently humoral information (SS) to neural information. How Does the Hepatic Receptor Mechanism?

2?

SOMATOSTATIN

Vagal Recognition

of SS Involve SS

The specificity of the neural chemoreception to intraportal SS appearance and its mechanism(s) are interesting. To determine whether the neural chemoreception involves the specific SS receptor mechanism, we modified the function of the SS receptor in the hepatoportal area. This became feasible by intraportally injecting a monoclonal antibody against rat neural SS receptor, which we raised.‘* The monoclonal antibody (MAb) of IgGl (k) isotype bound to the SS binding site of the receptor in competition with the ligand.‘* When 100 or 200 PL of the ascites containing the MAb against SS receptor was administered intraportally in the basal state, the afferent discharge rate in the hepatic vagus did not

FIG. 1. Afferent impulse discharges

before (a) and after (b) intraportal injection of somatostatin-14 (3.05 pmol) recorded in situ from hepatic branch of vagal nerve in rat (upper panel; a and b correspond, respec-

*

tively, to those recorded at the time in lower panel) and time course of the discharge rate in a rat (lower panel). Different rat in each trace. Each arrow shows the time of intraportal injection. SS, somatostatin14 (100 ~1); CA, control ascites (100 ~1); SSRA, ascites containing a monoclonal IgGl (k) antibody to SS receptor ( 100 ~1). (Reproduced

with permission from the .I Autonom Nerv Syst 1994;50:45 by copyright permission of Elsevier Science B.V.)

area has drawn much attention, little is known of the vagal chemoreception to GEP hormones in the portal circulation. SS-INDUCED CHANGES IN THE HEPATIC AFFERENT ACTIVITY

We examined whether the appearance of SS into the portal vein changes the afferent activity in the hepatic vagal nerve in anesthetized rats. Analysis of the activity was performed as described elsewhere.6*‘0 Briefly, the afferent nerve activity was measured in the nerve filaments isolated from the hepatic branch of the vagus under a dissection microscope. The distal cut end of the nerve filaments was placed on a pair of silver wire electrodes and the discharge rate of the firing spikes was measured in situ and analyzed. How Does Intraportal Appearance of SS Change Afferent Discharge Rate in the Hepatic Vagus? The injection of SS (at a dose of 3.05 pmol for 60 s) into the portal vein provoked a substantial increase in the hepatic vagal afferent activity within a few minutes after starting the

01 ,

0

I

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FIG. 2. Effect of intraportal injection of somatostatin-14 (3.05 pmol) (solid line, n = 5 ) and of administration of ascites containing a monoclonal IgGl (k) antibody to somatostatin receptor ( 100 ~1) 5 min before the somatostatin injection (broken line, II = 9) on rate of afferent impulse discharges in hepatic branch of rat vagal nerve. Error bars represent SEM. *P < 0.05 and * *P < 0.005 versus value before the injection, respectively. Arrow with SS shows the time of the somatostatin injection. (Reproduced from the J Autonom Nerv Sys 1994;50:45 with permission.)

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change for up to 180 min. as the discharge rate did not change ascites administration.” The results suggest that the hepatic vagus cannot recognize an unchanging level of SS at its low concentration. However, the intraportal administration of 100 PL of the MAb ascites 5 min before the intraportal SS injection (3.05 pmol) completely abolished the increase in the afferent discharge rate that otherwise occurred in response to the SS injection (Figs. 1 and 2) .I’ The abolishing effect of the MAb administered once into the portal vein lasted more than 120 min (Fig. 1). The control ascites (100 pL) administered intraportally 5 min before the SS injection did not exert any effect on the SSinduced changes of the discharge rate (Fig. 1). Furthermore. when 100 PL of the MAb ascites was administered 5 min after starting the SS injection, the SS-induced increase of the discharge rate disappeared in three out of five rats tested and diminished greatly in the other two. However, when the MAb ascites were administered 15 min after starting the SS injection in five rats or at 30 min in three rats, the MAb ascites of 100 PL or more up to 250 ,uL could not prevent the increasing discharge rate induced by the SS injection (Fig. 1). The results indicate that the intraportal appearance of SS facilitates the afferent activities in the hepatic vagus and that the antecedent intraportal administration of the MAb to SS receptor abolishes the SS-induced facilitation in the activities, suggesting that the receptor-mediated, neurochemoreceptive system monitors plasma SS levels in the portal vein. It is noteworthy that the effect of the MAb on the SS-induced changes of the afferent activity varies largely, depending on the timing of the antibody administration. The MAb used in this study inhibited the ligand binding to the receptor in vitro, regardless of its incubation with the receptor before the ligand binding.” Yet when the MAb was administered 15 min or more after the SS injection, the SS-induced effect on the discharge rate did appear, not accompanying any effect of the MAb (Fig. 1). A possible explanation for the results might relate to the levels of SS in the portal vein. If the concentration at 15 min or more after starting the SS injection had already been reduced to such a low level that the antibody did not exert any influence on the basal discharge rate, the effect of the antibody could not be observed. However, this seems to be unlikely, because the calculated SS levels at 15 min after cessation of the SS injection still remain above the basal concentration. Thus, the interesting results seem rather to emphasize the unique nature of the SSinduced change of the discharge rate: the change cannot be stopped by modulating the ligand binding to the receptor with the MAb, once a certain period of time (especially >5 min) has elapsed after the binding. This explanation seems to be pertinent to our observation that the afferent vagal activities lasted for such a long period of time-more than 120 min after a short-period SS injection of 60 s. The explanation may also be relevant to our previous observation: when a larger dose of SS was administered in the increasing phase of the activities induced by a forgoing SS injection, this additional, larger dose of SS failed to stimulate further increases in the pancreatic afferent vagal activity.” This might show an example of a desensitization-like phenomenon in the vagal recognition to SS, though the organ is different. Accordingly, it is intriguing to speculate that the present SS receptive system in the hepatoportal area has a property suitable for detecting the initial increase of intraportal SS concentration that is normally increased two to three fold for 3-4 h after a mixed meal. These observations represent the interesting characteristics of the vagal reception of SS in the hepatoportal area. Since five SS receptor subtypes (sstr l-5) that bind SS

on the control

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and/or SS-28 interesting to in the neural is recognized

selectively have been reported, I4 it is particularly know which subtype of SS receptor is involved SS recognition system and to what extent SS-28 by the system.

A NEURAL STRUCTURE RELEVANT TO THE CHEMORECEF’TION OF SS

To determine whether there is a morphological basis for this neural reception to SS in the hepatoportal area, we histologically examined the portal area of the liver in rats. AS shown in Figure 3, we eventually discovered the neural bodies that were located beneath the endothelium of the large branches of the rat portal vein. ” (The neural body had been termed the neural corpuscle in the report.) The bodies were ellipsoid (2040 pm in diameter) with their long axis along the bloodstream and protruding into the vessel lumen in various degrees. The body preferentially bound the exogenous SS injected into the portal vein as revealed by the immunostaining for SS.‘” As reported previously, “I we further observed the arborizations of rather thick nerve fibers in the body, resembling those known as the afferent apparatus of Krause, when the bodies were silverstained by the method of Holmes (Fig. 3, inset). To further examine whether SS receptors actually exist on the nerve terminals in the rat hepatoportal area, the area was immunostained by the labeled streptoavidin-biotin complex method using the monoclonal antibody against rat brain SS receptor.15 As shown in Figure 4A and reported previously by us, “I the encapsulated neural bodies (20-40 pm in diameter) were found at the beginning of the large branches of the portal vein 0.9- 1.5 mm apart from the porta of the liver.” By immunostaining with MAb to the SS receptor, fiber arborizations with terminal nodular swellings that looked like a structure of the afferent nerve endings, many in number, manifested themselves within the body (Fig. 4B and diagrammatically shown in Fig. 4C) .I5 Some of the arborizations were located just beneath the endothelium of the portal branch. The somewhat thick nerve fibers only near the terminal-like structures also showed the SS receptor staining in some sections of the body. No terminal arborizations or fibers with SS receptor stain-

FIG. 3. Three neural bodies (arrows) located beneath the endothelium of the large branch of the portal vein (P) of a rat, showing intense staining for somatostatin infused into the portal vein. Counterstain with hematoxylin. Inset shows the terminal nerve arborizations in the core of the body. Holmes’ stain. Bar, 100 /Im. (Reproduced from the Neurosci Lett 1986;67:78 by copyright permission of Elsevier Scientific Publishers Ireland Ltd.)

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ing were observed in the hepatic parenchyma. The sections stained with the antibody-free control ascites did not reveal either such fiber arborizations with terminal swellings or the

fibers. The results indicate that the SS receptor in the body is expressed almost exclusively on the tips of the last arborizations of nerve fibers. This adds further evidence to our previous observation that showed the hepatic vagal reception to intraportal SS and the SS-binding neural body as a relevant structure.“’ As to the fine terminal arborizations themselves in the neural bodies, the size and structure of the terminal-like structures quite resemble those of the fine terminal arborizations in the corpuscles within the glomus-like neurovascular body observed in the rat peripancreatic sinusoidal vein: we have reported that the neurovascular body is possibly relevant to the local neurochemoreception to SS.” However, in the aspect of SS-binding nature, the neural body in the hepatoportal area, as a whole, preferentially bound the exogenous SS injected into the portal vein, as evidenced by an outstanding diffuse SS staining.” On the other hand, only the terminal nerve structure in the corpuscle within the glomus-like body caught exogenous SS injected into the pancreatic artery. as clearly revealed by the immunostaining for SS.” Thus, it is intriguing that the neural body attached to the portal vein itself traps SS at a high blood flow, low SS concentration area, whereas the tiny nerve structure in the capillary-rich, “soaking up” glomus-like body preferentially catches SS at a low blood flow, high SS concentration region near the pancreas. The nature of the diffuse staining for SS in the neural body remains to be solved. In any event, the presence of the SS receptor on the tip of the terminal arborizations contained in the SS-trapping neural body could provide the efficient means for SS reception in the portal vein. The anatomical relation between the hepatic afferent vagus and the terminal arborizations expressing SS receptor in the neural body still remains to be clarified. However, the combined results of these immunohistological and electrophysiological studies strongly suggest that a peptide hormone secreted from the splanchnit organs acts on its own specific receptor on the afferent vagal nerve in the hepatoportal area. Indeed, the precise post-receptor mechanism in the afferent nerve terminals in the neural body”.” remains an important subject for future investigation. PHYSIOLOGICAL

FIG. 4. (A) The neural body (thick arrow) associated with the large branch (v) of the intrahepatic portal vein (P) Thin arrow indicates the neural body accompanied by the nerve bundle. H, hepatocytes; B, peribiliary glands. Bar, 100 pm. H-E staining. (B) A subendothelial neural body in the adjacent section (corresponding to the body indicated by the thick arrow in Fig. 4A) includes many fiber arborizations with the terminal nodular swellings. Immunostaining by labeled streptoavidin-biotin method using the ascites containing the monoclonal antibody to somatostatin receptor [IgGl(K), SRA-17E; 1:50 dilution]. Bar, 10 pm. (C) Diagram showing the fiber arborizations with terminal nodular swellings (arrows) in the neural body associated with the large branch (V ) of the intrahepatic portal vein seen in Fig. 4B. Bar, 10 pm. (Reproduced with permission from the Neurosci Lett 1995; 183:46.)

IMPLICATION OF THE SS-SENSING THE HEPATOPORTAL AREA

SYSTEM IN

The generation of the afferent signal through the vagal SS reception in the hepatoportal area involved the specific receptor and its unique postreceptor mechanism. Concerning the vagal reception to other GEP hormones, we have recently observed that the hepatic vagus is also receptive to one of the progluca gon-derived peptides: truncated form of glucagon-like peptide- 1(7-36) amide ( tGLP- 1) , known to have strong insulinotropic action as a physiological incretin, facilitated the hepatic vagal afferents when its physiological dose is injected into the portal vein.‘h,‘7 Moreover, full-length GLP-1 ( l-37), known to have no insulinotropic action, did not evoke any changes in the afferents even at larger doses.‘” The results indicate that the vagal chemoreception to tGLP-1 involves the specific receptor system for tGLP-1 in the hepatoportal area, which can distinguish the N-terminal difference of only six amino acids between tGLP-1 and GLP-1(1-37). In addition, another physiological incretin hormone, glucose-dependent insulinotropic polypeptide, did not change the hepatic afferents at various doses.” Taken together, it is conceivable that the hepatic vagus can recognize GEP hormones as far as the specific receptor for each of the GEP hormones is expressed on the afferent terminals of the nerve. It is interesting that tGLP-

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1 evoked a similar time course of the changes in the hepatic vagal afferent activity as observed in the case of SS. This observation implies that the neural recognition to both peptides involves a similar postreceptor signaling mechanism. In this regard, the question how the afferent information, which looks similar in the features of the electrophysiological events when the hepatic vagus senses different hormones (SS or tGLP-I), can be organized to exert different action in and/or via the central nervous system is important. Further studies are needed. It will be physiologically important if the afferent signal triggered by intraportal appearance of SS diverges onto the splanchnit organs via the vagal and/or the sympathetic nerve as efferent pathways. In fact, we have recently observed that SS administered into the portal vein suppresses the efferent spike activities in the small intestinal vagus and facilitates the efferents in the intestinal sympathetic nerve in rats (unpublished observation). These results seem to be pertinent to our previous observation showing that an intraportal SS administration attenuated the entry of fat from the intestine to blood circulation under the intact, but under disrupted, vagal (extrinsic) innervation.’ Thus, the neural monitoring system for SS (also for tGLP- 1) in the hepatoportal area has the potential ability to convey information to various parts of the central nervous system and to distribute a variety of information to the peripheral target organs.”

229

APPEARANCE SUMMARY

Our present results indicate that circulating SS in the portal vein generates signals to the central nervous system by converting humoral information, SS, to neural information through the unique receptor mechanism. This is the first observation providing substantial evidence that a peptide hormone secreted from the splanchnic organs acts on its own specific receptor on the afferent vagus in the hepatoportal area. Such a system could provide an efficient pathway for monitoring the endocrine states of the abdominal visceral organs, and contribute to regulation of appetite, eating behavior, and nutrient homeostasis. Those results also strongly suggest a role for the hepatic vagus as the afferent limb in reflex circuits via the central or peripheral nervous system. If so, the efferent parasympathetic and/or sympathetic limbs could provide effective means for controlling neurally and vascularly the endocrine and exocrine function of the splanchnic organs. ACKNOWLEDGEMENTS

The author thanks Akira Niijima, MD (Department of Physiology, Niigata University School of Medicine), and Yoriaki Kurata, MD (Department of Pathophysiology, Cancer Research Institute, Kanazawa University), for their support in accomplishing this work.

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10. Nakabayashi H, Niijima A, Kurata Y, Usukura N, Takeda R. Somatostatin-sensitive neural system in the liver. Neurosci Lett 1986;67:78 11. Nakabayashi H, Niijimd A, Nishizawa M, Nakabayashi IO, Takeda R. A unique receptor-mediated mechanism in vagal chemoreception of somatostatin in the hepatoportal area. J Auton Nerv Syst 1994; 50:45 12. Nakabayashi IO, Nakabayashi H. Monoclonal antibodies to somatostatin receptor of rat brain. Hybridoma 1992; 11:789 13. Nakabayashi H, Niijima A, Kurata Y, et al. Pancreatic vagal nerve is receptive to somatostatin in rats. Am J Physiol 1987;253:R200 14. Reisine T, Bell GI. Molecular biology of somatostatin receptors, Endocr Rev 1995; 16:427 15. Nakabayashi H, Kobayashi K. Nakabayashi IO, Kurata Y. Somatostatin receptor on the afferent nerve terminals in the rat hepatoportal area. Neurosci Lett 1995; 183:46 H, Nishizawa M, Nakagawa A, et al. Vagal hepato16. Nakabayashi pancreatic reflex effect evoked by intraportal appearance of tGLP1. Am J Physiol 1996;271:E808 17. Nishizawa M, Nakabayashi H, Uchida K, et al. The hepatic vagal nerve is receptive to incretin hormone glucagon-like peptide- I, but not to glucose-dependent insulinotropic polypeptide, in the portal vein. J Autonom Nerv Syst 1996;61:149