Elsevier

Regulatory Peptides

Volume 121, Issues 1–3, 15 September 2004, Pages 73-81
Regulatory Peptides

The rat pancreatic islets: a reliable tool to study islet responses to cholecystokinin receptor occupation

https://doi.org/10.1016/j.regpep.2004.04.017Get rights and content

Abstract

This study was undertaken to show that rat purified islets can be used as a reliable tool to study some aspects of human islet's physiology related to CCKR occupation. Therefore, isolated foetal, adult human and rat islets were compared for (1) CCKR subtypes mRNA and protein expression and somatostatin (SS) mRNA and (2) co-localization of these receptors with insulin, glucagon and SS. Finally, rat islets were tested for their responsiveness to stimulation. Purified human and rat islets were used for CCKR subtypes and SS mRNA estimation by RT-PCR and protein by Western blots. Receptors and hormones co-localizations were evaluated by confocal microscopy. Hormones secretion served to determine rat islets responsiveness. Islets of both species express CCKA and CCKBR mRNA and proteins and SS mRNA. The CCKAR co-localizes with insulin and glucagon and the CCKBR with SS. Insulin release was increased 5-fold in response to 16 mM glucose and SS secretion reached 1.3- and 1.7-fold increments above basal in response to forskolin and IBMX.

In conclusions, human and rat islets have comparable CCKR subtypes localized on the same cells; they also express SS mRNA. The rat islets are functional as they secrete but their response to hormonal stimulation remains to be clarified. These rat islets can thus serve as tools to study islets physiology.

Introduction

Since the molecular cloning of the rat CCKA and human CCKB receptors [1], [2], numerous studies were published on the expression of these two receptor subtypes in the pancreas of different species. By Northern blot analyses, the CCKAR was identified in rat [1] and mouse [3] pancreas. Using the RT-PCR technique, a more sensitive method to detect lower abundance transcripts, the expression of CCKAR was also detected in rat pancreatic islets [4] and human pancreas [5].

The presence of the CCKBR in rodent pancreas has been controversial. Indeed, in rat and mouse, many investigators failed to detect its mRNA by Northern hybridization [3], [6], [7], [8]. However, its presence was confirmed by RT-PCR in both species [3,4,8]. The human pancreas seems to have more abundant levels of CCKBR than CCKAR as determined by RT-PCR [9]; this observation may explain why the CCKBR was much easier to detect in this species by Northern blot analysis [1], [5], [10].

With the development of specific antibodies raised against the CCKA and CCKB receptors, it became possible to characterize the proteins of these two receptors as well as their localization on the various cells of the endocrine and exocrine pancreas. By immuno-fluorescence, the CCKAR was found on acinar cells of the rat and mouse pancreas but absent from those of the pig and human pancreas. This receptor was also found on rat, mouse and pig pancreatic beta cells [11]. By immunohistology, the CCKAR appeared on porcine pancreatic alpha cells [12], a site different from that of two other studies locating the CCKBR on human [13] and rat [14] pancreatic alpha cells; adding to the confusion, our immunofluorescence data localized the CCKB receptor on pancreatic delta cells of the rat, mouse, pig, human [8] and calf pancreas [15].

Our initial observations indicated that the CCKBR was constantly associated with the somatostatin delta cells in rat, mouse, human, pig, calf, dog and horse pancreas [8], [16]. These data were obtained from freshly frozen pieces of pancreatic tissue in liquid nitrogen, of which frozen sections were made; the biochemical characterization of this CCKBR protein from several species was always performed out of membranes prepared from whole tissue, including exocrine and endocrine glands.

As a result of this specific location of the pancreatic CCKBR protein on the somatostatin delta cells, the challenging question was to find a functional role for gastrin through occupation of its CCKB receptor on these cells. Our hypothesis stipulates that gastrin could be involved in secretion and/or synthesis of somatostatin as well as in the growth control of the delta cells.

Access to fetal and adult human pancreatic tissue is rather difficult and often very occasional in specialized centers devoted to abortion or organ transplantation. The human pancreatic component of this study was introduced to demonstrate that the human and rat pancreatic islets are quite comparable with regards to CCK receptor subtypes mRNAs and protein expression and localization so that they can be used indifferently wherever available to study islet physiology related to CCK receptors' occupation.

Section snippets

Experimental subjects and animals

Human pancreatic tissues were obtained from normal elective pregnancy terminations (fetus) or organ donors (adult). These studies were approved by the Institutional Human Subject Review Boards. Animals: Male Sprague–Dawley rats (340–360 g) were purchased from Charles River Laboratories, St. Constant, QC. Before sacrifice, they were housed in a light and humidity controlled room and given free access to food and water; they were fed when sacrificed. These animals were used according to our

Tissue preparations

Once excised, human fetal and adult, and rat adult pancreas were quickly frozen in liquid nitrogen and kept frozen at −80 °C until they were processed either for Western blot analysis or RNA extraction.

Rat islets isolation

After anesthesia with Somnotol (MTC Pharmaceutical, Cambridge, Ontario), the pancreatic islets were isolated using the collagenase digestion method of Lacy and Kostianovsky [17] as recently extensively described [18].

Human fetal islets isolation

The fetal pancreas was washed in HBSS and cut in fine pieces with scissors in

Evaluation of CCK receptor mRNAs by RT-PCR

With the RT-PCR technique, the CCKAR mRNAs expression presents two transcripts in adult and fetal human purified islets as well as in rat islets and whole pancreatic gland (Fig. 1A, CCKAR).

The same sensitive technique indicates that mRNAs isolated from adult and fetal human and rat purified islets and rat total pancreatic gland also exhibit one transcript of the CCKBR (Fig. 1A, CCKBR). In both species, levels of the CCKBR mRNAs are much less abundant than those of the CCKAR.

The somatostatin

Discussion

This study was designed to demonstrate that the rat purified pancreatic islets can be used as a useful tool to study some aspects of human islet physiology related to CCK receptors' occupation. Our data clearly indicate for the first time that (1) islets purified from fetal and adult human and rat pancreas express identical transcripts of CCKAR mRNAs; (2) the fetal and adult human islets also exhibit a single CCKBR mRNA transcript as in rat islets; (3) the somatostatin mRNAs are identically

Acknowledgements

We thank Mrs. Christiane Gauvin for secretarial assistance, Dr. Abou Elela and Mr. Mathieu Catala for use of their confocal microscope. We also thank Professor Bernard P. Roques from the Université René Descartes (Paris V) for his supply of pBC264. This research was supported by grant GP6369 from the Natural Sciences and Engineering Research Council of Canada.

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