" Active Transport of Calcium in Inverted Vesicles of Escherichia coli

A discrete population of cells of the endocrine pancreas contains immunoreactive somatostatin as shown by immunofluorescence. These cells are different from those containing glucagon or insulin. This unexpected observation may be of physiopathological significance in the regulatory mechanisms involved in the secretion of glucagon and insulin. Somatostatin is an oligopeptide with the primary sequence isolated from ovine hypothalamic extracts (1) on the basis of its activity to inhibit the secretion of adenohypophysial growth hormone (2, 3). Availability of the peptide in large quantities obtained by total synthesis (4) has shown it to be biologically active, both in the cyclized (oxidized) or linear (reduced) form, to inhibit the secretion of growth hormone in all the species studied so far. Recently, it has been shown that somatostatin also inhibits the secretion of glucagon and insulin (5) by acting directly at the level of the cells of the endocrine pancreas (5-7). Furthermore, on the basis of bioassays, somatostatin or somatostatin-like substances have been shown to have a large extra-hypothalamic distribution in the central nervous system (8). This set of observations, combined with the evidence of a very short (<4 min) biological half-life for somatostatin upon intravenous injection, led to the hypothesis that the peptide might be delivered to the endocrine cells of the pancreas by peripheral nerves with peptidergic endings. This was explored by immunohistochemistry, a method which had previously led to localize (immunoreactive) somatostatin in the median eminence (9-11) as well as in discrete nerve fibers in the pars tuberalis (11). The results reported here* will show that, contrary to expectations, no nerve fibers oi endings containing immunoreactive somatostatin were found in the pancreas of the several species studied; unexpectedly, however, a definite population of cells in the islets of Langerhans was found to contain immunoreactive somatostatin. MATERIALS AND METHODS (a) Production of Antisera to Somatostatin. Somatostatin and its reduced form, H2 somatostatin, were coupled to human serum albumin with glutaraldehyde or bisdiazotized benzidine. The complexed antigen was injected with adjuvant into rabbits. A detailed description of the immunization procedure has been published previously (11). Only animals injected with the oxidized form of somatostatin yielded detectable antibodies. The antiserum used here is our lot no. 1251. (b) Other Antisera. Well-characterized antisera against polypeptides known or suspected to be present in the endocrine pancreas were obtained as follows: An antiserum to pancreatic glucagon (ref. no. GB 5667, courtesy of Dr. R. Assan, Hdtel Dieu, Paris); an antiserum to (bovine) insulin (ref. no. AIS III, courtesy of Dr. R. Unger, Dallas, Texas); an antiserum to gastrin (ref. no. 11, courtesy of Dr. W. Gepts, Brussels). (c) Polypeptides. The following polypeptides were obtained in highly purified form and were used to ascertain the specificity of the various immunoreactions utilized here: oxytocin, [Arg8]vasopressin, neurophysin-A, luteinizing hormone releasing factor (LRF) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NH2), thyrotropin releasing factor (TRF) (pGluHis-Pro-NH2), insulin, glucagon, secretin (Karolinska Institute G1H Research unit, lot no. 17281, courtesy of Dr. A. Renold, Geneva), somatostatin, H2somatostatin, the tetrapeptide H-Thr-Phe-Thr-Ser-OH (the latter three peptides synthesized by Dr. J. Rivier, the Salk Institute, La Jolla, Calif.); this tetrapeptide represents a sequence common to somatostatin (Thr'0 ... Ser'3), glucagon (Thr. ... Ser8) and secretin (Thr5... Ser8). (d) Tissues Samples. Fragments of or whole pancreas were obtained from the following species: Man, sheep, ox, pig, rat, chicken. For control studies, the following tissues were obtained from rats, sheep, and pigs: (1) liver, gallbladder, salivary glands, stomach, duodenum; (2) kidney, urinary bladder; (3) testes, epididymis, vas deferens, seminal vesicles, prostate; (4) thymus, spleen, lymph nodes; (5) pineal body, Gasser's ganglion; (6) thyroid. All tissues were fixed for 2-4 days in Bouin-Hollande fluid free of acetic acid and added with 10% saturated Hg-sublimate; they were then thoroughly washed in water, dehydrated, and included in paraffin. Sections (5Mm) were used after affixing on glass slides with 1%