Remarkable adrenergic nerves in the exocrine pancreas

Summary The adrenergic nerves in the pancreas of mice, rats, guinea-pigs, rabbits, and cats were investigated with the fluorescence method of Falck and Hillarp. The relations between the adrenergic fibres and the vessels were studied by the injection of india ink into the vessels.Besides the normal manifestation of adrenergic fibres at the large vessels, some vessels of capillary size were also accompanied by adrenergic fibres. These fibres had a very weak fluorescence, and showed up regularly only when the animal had been treated with Nialamide and L-DOPA or dopamine to increase the catecholamine content of the adrenergic fibres. The weakness of the fluorescence is perhaps due to low transmitter concentration or to small size of the nerve fibres, or to both. A rough estimate indicated that either the transmitter concentration of the nerve fibre is at least approximately 100 times below that seen in adrenergic nerves in other tissues, or that the radius of the varicosities of the nerve fibres is less than 0.2 . Neither alternative has previously been recognized.The secretory acini of the pancreas seem to lack a direct adrenergic supply. In the intrapancreatic ganglia, non-fluorescent nerve cells were reached by adrenergic terminals. No adrenergic nerve cells were detected in the pancreas of rats and cats. Small intensely fluorescent catecholamine-containing cells were observed in connexion with the intrapancreatic ganglia of rats.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant NB 06701-01) by the Swedish Medical Research Council (project B 67-12X-712-02A), and by the Faculty of Medicine, University of Lund, Sweden.     

Ultrastructural localization of pancreatic polypeptide in the F cell of the dog pancreas.

The F cell of the dog pancreas has been identified as the specific cell type containing pancreatic polypeptide. This localization of pnacreatic polypeptide was accomplished by immunocytochemical staining of ultrathin sections and direct electron microscopic identification. Verification of the specificity of the reaction was obtained by blocking experiments on serial sections of the same cell. It is proposed that the name F cell be used for defining in all species the islet cell that contains pancreatic polypeptide.     

Sympathoadrenal system in neuroendocrine control of glucose: mechanisms involved in the liver, pancreas, and adrenal gland under hemorrhagic and hypoglycemic stress.

Glucose homeostasis is maintained by complex neuroendocrine control mechanisms, involving three peripheral organs: the liver, pancreas, and adrenal gland, all of which are under control of the autonomic nervous system. During the past decade, abundant results from various studies on neuroendocrine control of glucose have been accumulated. The principal objective of this review is to provide overviews of basic adrenergic mechanisms closely related to glucose control in the three peripheral organs, and then to discuss the integrated glucoregulatory mechanisms in hemorrhage-induced hypotension and insulin-induced hypoglycemia with special reference to sympathoadrenal control mechanisms. The liver is richly innervated by sympathetic and parasympathetic nerves. The functional implication in glucoregulation of sympathetic nerves has been well-documented, while that of parasympathetic nerves remains less understood. More recently, hepatic glucoreceptors have been postulated to be coupled with capsaicin-sensitive afferent nerves, conveying sensory signals of blood glucose concentration to the central nervous system. The pancreas is also richly supplied by the autonomic nervous system. Besides the well documented adrenergic and cholinergic mechanisms, the potential implication of peptidergic neurotransmission by neuropeptide Y and neuromodulation by galanin has recently been postulated in the endocrine secretory function. Presynaptic interactions of these putative peptidergic neurotransmitters with the classic transmitters, noradrenaline and acetylcholine, in the pancreas remain to be clarified. It may be of particular interest that it was vagus nerve stimulation that caused a dominant release of neuropeptide Y over that caused by sympathetic nerve stimulation in the pig pancreas. The adrenal medulla receives its main nerve supply from the greater and lesser splanchnic nerves. Adrenal medullary catecholamine secretion appears to be regulated by three distinct local mechanisms: adrenoceptor-mediated, dihydropyridine-sensitive Ca2+ channel-mediated, and capsaicin-sensitive sensory nerve-mediated mechanisms. In response to hemorrhagic hypotension and insulin-induced hypoglycemia, the sympathoadrenal system is activated resulting in increases of adrenal catecholamine and pancreatic glucagon secretions, both of which are significantly implicated in glucoregulatory mechanisms. An increase in sympathetic nerve activity occurs in the liver during hemorrhagic hypotension and is also likely to occur in the pancreas in response to insulin-induced hypoglycemia. The functional implication of hepatic and central glucoreceptors has been suggested in the increased secretion of glucose counterregulatory hormones, particularly catecholamines and glucagon.     

Development of the pancreas in medaka

In the present work, pancreatic organogenesis has been studied in the medaka (Oryzias latipes), a teleost fish with several advantages as an experimental system in developmental biology. We demonstrated that the pancreas develops from three primordia budding from the dorsal and ventral faces of the gut epithelium. Such buds then fuse to form a single endocrine islet surrounded by exocrine tissue. Interestingly, the endocrine tissue forms only from the dorsal bud. We next analyzed a collection of medakas that had been hybridized with cDNAs derived from an anterior brain library. We found new clones expressed in the pancreatic region demonstrating that the medaka can be used to define new genes expressed in the pancreatic region that follow a specific spatial and temporal pattern of expression.     

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.     

Ductal cells of the pancreas

Ductal cells of the pancreas form the epithelial lining of the branched tubes that deliver enzymes produced by pancreatic acinar cells into the duodenum. In addition, these cells secrete bicarbonate that neutralizes stomach acidity. During development, epithelium of endodermal origin evaginates from the future duodenum area and invades the mesenchyme to form a complex branched network. All endocrine, acinar and ductal cells arise from common precursors in this epithelial structure. Adult ductal cells share some similarities with embryonic primitive ducts and may retain the ability to generate endocrine cells in the adult. Based on challenged pancreas regeneration experiments, the adult ductal cells have been proposed to be pancreatic stem cells but their role in normal endocrine cell turnover has recently been challenged. Manipulating their ability to give rise to endocrine cells may open new avenues in the treatment of diabetes and therefore they have recently been under scrutiny. In addition, in the main form of pancreatic cancer, pancreas adenocarcinoma, tumor cells share similarities with ductal cells. The secrets of an appropriate therapy for this deadly cancer may thus reside in the biology of ductal cells.     

Galanin and the endocrine pancreas

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic beta-cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated beta-cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage-activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization-induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

Morphology of the endocrine part of the pancreas of birds]

Methods of light and electron microscopy were used to study the structural organization of the endocrinous part of the pancreas in birds (domestic ducks and chickens). It has been established that the endocrinous part is formed by three types of pancreatic islands: dark, light and mixed islands. "Dark" islands consist of A- and D-cells, "light" ones--of B and D-cells and "mixed" islands--of A-, B- and D-cells. The index of B/A-cell ratio is equal to 0,39--0,4. This shows that each B-cell corresponds to 4 A-cells. On this basis one can say that the counterinsular apparatus of birds became stronger in the process of evolution and due to this a comparatively high level of glycemia is sustained in them. Availability of "dark" and "light" pancreatic islands also points out that in birds there occurred a partial disconnection of insular and counterinsular components of the endocrinous part of the pancreas, necessary for adaptation to principally new conditions of the environment.     

Central adrenergic suppression augments the insulin and glucagon secretory, and the glycogenolytic responses in streptozotocin-diabetic rats.

It has been suggested that the increased activity of the sympathetic nervous system and the resultant increase in the tissue catecholamine levels contribute to the pathogenesis of diabetes. In this study we evaluated the effect of clonidine, a central adrenergic agonist that decreases sympathetic tone, on the serum levels of glucose, insulin, glucagon and norepinephrine and on the hepatic glycogen content in normal and streptozotocin-diabetic rats. The animals were treated with clonidine 25 micrograms/kg/day interperitoneally for 3 weeks to suppress the central adrenergic impulses. Clonidine treatment significantly increased the weight gain, but did not affect plasma glucose, insulin, glucagon and norepinephrine in the diabetic animals. Pancreatic insulin and liver glycogen contents were significantly higher in the clonidine-treated than in the untreated diabetic rats. However, clonidine did not affect pancreatic insulin and liver glycogen content of nondiabetic animals. The intravenous administration of glucagon increased plasma glucose in the clonidine-treated, but not in the saline-treated diabetic rats. Insulin-induced hypoglycemia significantly enhanced glucagon release in clonidine-treated but not in saline-treated diabetic rats. We conclude that the suppression of central adrenergic activity may ameliorate the effects of insulin insufficiency on pancreatic hormone secretion and hepatic glycogen content.     

Effects of endothelin on microcirculation of the pancreas.

Endothelin, a newly described endothelial-derived peptide, has potent vasoconstrictive properties and has been speculated to play a physiological role in the regulation of blood flow in some organs. The present study was designed to evaluate the effects of endothelin-1, endothelin-2 and endothelin-3 on the pancreatic microcirculation. Pancreatic tissue blood flow was measured by a laser Doppler flow meter in anesthetized dogs and endothelin-1, endothelin-2 or endothelin-3 was injected intravenously in graduated doses. Endothelins induced dose-dependent decreases in pancreatic tissue blood flow. Endothelin-1, endothelin-2 and endothelin-3 at a dose of 100 pmol/kg reduced pancreatic blood flow by 45.4%, 19.6% and 51.9%, respectively, whereas systemic arterial blood pressure was not significantly affected. When endothelin-3 was administered at a dose of 1000 pmol/kg, pancreatic blood flow was decreased by 73.5% with a concomitant increase of systemic arterial blood pressure by 17.6%. Endothelins potently decreased pancreatic tissue blood flow, suggesting a possible role of these agents in regulating the pancreatic microcirculation.     

Formation of protein in the pancreas

1. The total known secretory enzyme content of the mouse pancreas has been determined and found to represent about 20 per cent of the weight of the dry, fat-free tissue. 2. The changes in secretory enzyme content that occur during the cycle of secretion and synthesis have been measured. 3. In the course of the cycle no significant changes have been found in DNA or RNA content of pancreatic tissue. 4. Constancy of DNA content, along with other observations, indicates that total protein content of the gland remains substantially unchanged during the cycle of secretion and synthesis. These facts point to the conclusion that following upon the secretion of enzyme protein, synthesis of new protein takes place relatively rapidly in the exocrine cells of the pancreas and this protein is then gradually transformed into the characteristic pancreatic enzyme proteins.     

Early indicators of exocrine pancreas carcinogenesis produced by non-genotoxic agents

In the past 40 years the incidence of pancreatic cancer in many Western countries had increased. Since no single factor responsible for the development of pancreatic cancer has been identified, it is believed that non-genotoxic factors may play an important role in the pathogenesis of this highly fatal form of cancer. Focal abnormalities of acinar cells, referred to as atypical acinar cell foci or nodules, occur spontaneously in rats and some other species. Their incidence increases with age from zero at birth to about 75% in 2-year-old rats. These spontaneous lesions have a phenotype that cannot be distinguished from the putative, atypical preneoplastic, acinar cell foci induced in rat pancreas by the carcinogen azaserine. Unsaturated fat (corn oil) has been found to increase the incidence of atypical acinar cell nodules and adenomas in the pancreas of non-carcinogen-treated rats without influencing the weight of the pancreas. Furthermore, unsaturated fat has a specific promoting effect on the growth potential of atypical acinar cell foci and nodules induced in rat pancreas by azaserine, resulting in an increase in the number and size of these lesions. Rats fed raw soya flour or trypsin inhibitors develop an enlarged pancreas as a result of hypertrophy and hyperplasia. They also develop acidophilic atypical acinar cell foci and nodules, adenomas and adenocarcinomas after being fed full-fat raw soya flour for 2 years. It may be concluded from the observations in rat pancreas that non-genotoxic compounds or conditions that enhance pancreatic growth may be classified as non-genotoxic pancreatic tumour promoters. The observations with corn oil, however, indicate that there may be non-genotoxic compounds that specifically enhance growth of spontaneous initiated atypical acinar cell foci without causing hyperplasia of the pancreas. The possible mechanisms whereby unsaturated fat and trypsin inhibitors exert their effects on exocrine pancreatic carcinogenesis are discussed.     

Cellular localization of intrinsic factor in pancreas and stomach of the dog

Summary A cobalamin (vitamin B₁₂)-binding protein has recently been identified in canine pancreatic juice which is biochemically, immunochemically and functionally similar to canine gastric intrinsic factor. However, the cellular sources of both this pancreatic intrinsic factor and gastric intrinsic factor in the dog are not known. Antisera raised against canine gastric intrinsic factor have been used to examine the distribution of intrinsic factors in the canine pancreas and stomach. Immunoreactivity was demonstrated in duct cells but not acinar or endocrine cells in the pancreas, and in fundic peptic and pyloric gastric pit cells in stomach. All immunostaining was abolished by preabsorption of the antisera with purified canine gastric and pancreatic intrinsic factors. A cellular source of pancreatic intrinsic factor has not been previously described, and the demonstration of intrinsic factor-like immunoreactivity in two cell types in the canine stomach contrasts with its localization in a single cell type in the gastric mucosa of other mammalian species. Furthermore, immunoreactivity in pancreatic duct cells was detected at much higher dilutions of antisera than those required for staining of peptic and gastric pit cells. This suggests a higher concentration of antigen, and supports previous evidence that the pancreas is a major source of intrinsic factor in the dog.     

Purification of classical pancreatic lipase from dog pancreas

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.     

Typing of the alpha adrenergic receptors of pancreatic B cells. In vitro studies with the isolated and perfused rat pancreas

Two groups of receptors, one which develops a stimulating effect (alpha 1), the other an inhibitory effect hae recently been isolated in the alpha adrenergic group. In order to type the B cell adrenergic receptor of the endocrine pancreas, which mediates the inhibitory action exerted by the catecholamines on insulin secretion, the release of this hormone was evaluated in the presence of five alpha simpathomimetic substances that have a decreasing degree of efficiency on the adrenergic alpha 2 receptor of the presynaptic sympathic nerve terminal. The order of potency with which the alpha agonists tested depressed IRI secretion is superimposable on that of their potency on the sympathetic nerve and alpha 2 receptor. We concluded that adrenergic inhibition of insulin secretion is mediated by an alpha 2 receptor.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Ouabain- and potassium-induced stimulation of amylase release in fragments and acini of rabbit pancreas

Ouabain increases the enzyme secretion from the isolated rabbit pancreas and pancreatic fragments, but not from isolated pancreatic acini. The increase occurs after a delay of 45-60 min and is not accompanied by an increase in lactate dehydrogenase release. The stimulatory effect of ouabain (10(-5) M) is dependent on the presence of extracellular calcium, and is not antagonized by 10(-4) M atropin, 10(-4) M propranolol, 10(-5) M phentolamine, 10(-3) M dibutyryl-cyclic GMP, 10(-6) M tetrodotoxin, 10(-4) M verapamil or 10(-4) M D-600. Elevation of the extracellular potassium concentration to 120 mM in the presence of 10(-4) M atropin also increases the enzyme secretion from rabbit pancreatic fragments. The increase is again dependent on the presence of extracellular calcium and is resistant to adrenergic blockade and to tetrodotoxin, verapamil or D-600. Forskolin also stimulates a Ca2+-dependent release of amylase from pancreatic fragments but not from pancreatic acini. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IMX), ouabain (10(-5) M) and K+ (120 mM) cause an immediate increase in the cyclic AMP content of pancreatic fragments which does not occur in the absence of extracellular calcium. In pancreatic acini, the cAMP production is only slightly increased by ouabain. In the absence of IMX, the cAMP levels in fragments or acini are not detectably altered by ouabain or K+. The results suggest that the stimulation of enzyme secretion by ouabain and high K+ is an indirect effect, mediated by the release of an endogenous transmitter from non-cholinergic, non-adrenergic nerves in the intact preparations. The release and/or the effect of the transmitter appears to be mediated primarily by Ca2+ and secondarily by cyclic AMP.     

Blood vessels of the pancreatic islets in different portions of the adult human pancreas]

The pancreatic islets and their blood vessels have been studied in the head, the body and the tail of the human pancreas. The following methods have been applied: injection, histological and quantitative estimation, graphic and plastic reconstruction. A rather great variability in the form of the pancreatic islets has been stated, with presence of one--two peculiar processes in large islets. In different parts of the pancreatic gland, relative volume of the endocrine parenchyma has been stated to be statistically greater (2.16 +/- 0.45%) in the caudal portion than in the head of the gland (1.31 +/- 0.26%). In every pancreatic islet an afferent arterial vessel is described, two types of its branching are determined: magistral and scattered. Relative volume of the pancreatic islets and morpho-functional coefficient reflecting the ratio of the capillary surface area to the volume of the islet capillaries in different parts of the pancreatic gland have been estimated.     

Nervous system regulation of exocrine pancreatic secretion in the chicken

Acute assays were carried out using broiler chickens in which a reentry catheter had previously been placed chronically in the main pancreatic duct. Samples of pancreatic juice were collected after manoeuvres of blockage and stimulation with different neurotransmitters and blocking agents, both cholinergic and adrenergic, as well as electrical stimulation of the left vagosympathetic trunk. Stimulation of the vagus nerve induced a marked increase in the pancreatic flow but with no changes in the enzyme content. Acetylcholine was seen to cause a slight but significant increase in pancreatic flow and a non-significant increase in amylase activity. The drop in the flow of pancreatic juice in response to adrenaline was not very sensitive to adrenergic blockers. The effect of adrenaline on pancreatic secretion cannot be attributed to vascular changes.