Maurizio Bersani

Human galanin: Primary structure and indentification of two molecular forms

From acid/ethanol extracts of surgical specimens of human large intestine we isolated two peptides, in approximately equal amounts, that reacted with an antiserum against porcine galanin. By amino acid analysis, sequence analysis and mass spectrometry, the larger of the two peptides was found to consist of 30 amino acid residues, the sequence of which was identical to that of porcine galanin except for the following substitutions: Val16, Asn17, Asn36, Thr20 and Scr30. Unlike porcine galanin, the caboxy‐terminus was not amidated. The smaller peptide corresponded to the first 19 amino acid residues counted from the N‐terminus of the 30 residue peptide (again without amidation). The structural analysis was repeated on another batch of tissue with identical results. By HPLC analysis of extracts of specimens from a further 4 patients, the same peptides were identified. Thus, human galanin includes two peptides of 19 and 30 amino acids that share the sequence of the N‐terminal 15 residues with other mammalian galanins, but exhibit characteristic differences in the remaining part of the molecules.

Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy.

Dorsal root ganglia (DRG) neuronopathy was induced in rats by chronic treatment (2 mg/kg twice a week for nine injections) with the antineoplastic drug cisplatin. Morphological alterations and changes in peptide [calcitonin gene-related peptide (CGRP), substance P, galanin (Gal), and somatostatin] concentration were studied in the DRG, the spinal cord, and the sciatic nerve. Peptide concentration was increased in DRG neurons, with CGRP and Gal showing the highest increase. Conversely, in the sciatic nerve there was a general decrease in peptide content. In DRG a reduction in the nuclear, cytoplasmic, and nucleolar areas of primary sensory neurons was evident and was accompanied by clear-cut aspects of nucleolar structural damage. In peripheral nerves only extensive morphometric determinations could evidence a reduction in nerve conduction velocities and impairment in pain detection and coordination. Some of the nerve fibers presented axonal and adaxonal accumulations, suggesting the presence of an axonopathy. These results confirm that DRG cells are the primary target of cisplatin-induced neurotoxicity. Milder alterations can be detected in peripheral nerves. The increase in peptide concentration in DRG is probably due to cisplatin-related damage to the axonal transport system rather than to an increased synthesis.

Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract

Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C-terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the procine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, beta- and/or gamma-preprotachykinin, in the same neurons.

On the effects of human galanin in man

SummaryHuman galanin was recently isolated and sequenced and was found to differ from porcine galanin, hitherto used for studies in humans, in several important respects. We therefore synthesized and purified human galanin and infused it i.v. at a rate of 74 pmol · kg−1· min−1 into six healthy volunteers for 60 min during a hyperglycaemic clamp. The clamp was achieved by i. v. infusion of glucose at a rate which in a control experiment had been demonstrated to maintain the plasma glucose level at 12–13 mmol/l for 90 min. Galanin concentrations reached a plateau of approximately 1500 pmol/l throughout the infusion as opposed to pre-infusion and control levels of 20–30 pmol/l. The glucose levels obtained in the two experiments were indistinguishable. Plasma levels of C-peptide and insulin increased significantly in both experiments and the dynamic concentration curves were almost identical. Glucagon concentrations in plasma decreased significantly and similarly. Growth hormone levels, however, increased eight-fold during galanin infusions. Galanin was eliminated from plasma with a half-life of 3.7±0.4 min, similar to that of porcine galanin. It is concluded that human galanin powerfully stimulates growth hormone secretion in man, but has no effect on pancreatic endocrine secretion or glucose metabolism in the concentrations obtained in this study.

Somatostatin and prosomatostatin in the retina of the rat: An immunohistochemical, in-situ hybridization, and chromatographic study

Specific antisera, raised in rabbits, against somatostatin 1-14, somatostatin 1-28, the fragment 1-12 of somatostatin 1-28, and prosomatostatin 20-36 were used for immunohistochemistry and gel filtration of the rat retina. With all antisera, immunoreactive perikarya could be located in the inner nuclear and ganglion cell layers. In the inner nuclear layer, amacrine cells with processes extending predominantly into the first sublayer of the inner plexiform layer were observed. Some processes extended also to the ganglion cell layer. In addition, somatostatin-immunoreactive interplexiform cells were present in the inner nuclear layer. In the ganglion cell layer, perikarya were found located in the midperiphery and in the far periphery of the retina. The neurons located in the midperiphery of the retina possessed a round perikaryon from which processes could be followed going into the inner plexiform layer, where they dichotomized in the third and first sublayers. The perikarya in the far periphery of the retina near the ora serrata exhibited an ovoid-shaped cell body from which processes extended horizontally in a bipolar manner in the layer itself. By use of an [35S]-labeled antisense oligonucleotide probe, in situ hybridization of the rat retina showed the presence of perikarya in the inner nuclear layer and ganglion cell layer containing mRNA encoding for prosomatostatin. Gel filtration of the retinal extracts followed by radioimmunoassay showed the presence of somatostatin 1-14, the fragment 1-12 of somatostatin 1-28, and prosomatostatin 1-64. However, somatostatin 1-28 was not detected. The results obtained in this study verify the presence of somatostatin 1-14 in the rat retina located in perikarya and processes in the inner nuclear and ganglion cell layers. The positive in-situ hybridization signals show that the intraneuronal somatostatin immunoreactivity is due to synthesis of the peptide and not uptake in the neurons. The presence of the somatostatin propeptide and fragments of this propeptide, in both intraretinal perikarya and fibers, indicate a posttranslational modification of this neuropeptide in the perikarya and the processes as well.

Extrinsic control of the release of galanin and VIP from intrinsic nerves of isolated, perfused, porcine ileum

By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.

Isolation and Molecular Characterization of Porcine Calcitonin Gene-Related Peptide (CGRP) and Its Endocrine Effects in the Porcine Pancreas

The aim of this study was to investigate the possible role of porcine calcitonin gene-related peptide (CGRP) in the regulation of the endocrine porcine pancreas. Initially, we isolated and purified CGRP from extracts of porcine adrenal glands and pancreases. A single molecular form of the peptide was found in the two tissues. The adrenal peptide was sequenced and found to differ from human α-CGRP at six positions and from human β-CGRP at three positions. By immunohistochemistry, CGRP was found in nerve fibers in the pancreatic ganglia. A synthetic replica of the porcine peptide was infused at different dose levels (10−10, 10−9, and 10−8 M) into isolated perfused porcine pancreata. With 5 mmol/L glucose in the perfusate, CGRP at 10−10 and 10−9 M increased insulin and glucagon secretion, whereas significant decreases were observed with 10−8 M. Somatostatin secretion was increased significantly by 10−1 M CGRP. In immunoneutralization studies (n = 6) using a high-affinity somatostatin antibody, the inhibitory effect of CGRP at 10−8 M was reversed to a significant stimulation of insulin and glucagon secretion. Insulin secretion in response to square-wave increases in glucose concentration to 11 mM was inhibited dose dependently by CGRP; at 10−8 M the insulin output decreased by 72 ± 9% (n = 6). The present results indicate that CGRP may be involved in the regulation of insulin and glucagon secretion from the porcine pancreas.

Pigs produce only a single form of CGRP, part of which is processed to N- and C-terminal fragments.

Using radioimmunoassays with two different antisera, one directed towards the C-terminal and one towards the mid part of porcine and human alpha-CGRP, respectively, we isolated three immunoreactive peptides from acid/ethanol extracts of porcine spinal cord by means of HPLC. By amino acid sequence analysis and mass spectrometry (PDMS), the most abundant peptide was found to be identical to the 37 residue CGRP previously isolated from porcine adrenal glands and spinal cord. The two remaining peptides were identified as pCGRP(18-37) and pCGRP(19-37). Furthermore, the oxidized forms (oxidized Met in position 22) of all three peptides were isolated. We extracted a large amount of tissue and the extractable peptides were purified without discarding side fractions. The purification steps were monitored by immunochemical methods that are highly sensitive for human alpha- and beta-CGRP. Yet we were unable to detect any second full-length form of CGRP. Thus, we conclude that only a single form of full-length CGRP is found in pigs and that this peptide may be cleaved to produce potentially bioactive N- and C-terminal fragments.

Processing and Secretion of Prosomatostatin by the Pig Pancreas

Antisera and radioimmunoassays against five different regions of prosomatostatin (proSS) were used for chromatographical analysis and for immunohistochemical mapping of the products of proSS in the pig pancreas. Secreted products of proSS were studied by analysis of effluent from isolated perfused pig pancreas obtained during isoproterenol stimulation. All cells that were stained with one antiserum also stained with the other antisera. Immunoreactive nerves were not observed. Isoproterenol increased equally the secretion of proSS 20–36, proSS 65–76, and proSS 7%92 immunoreactivity. The major molecular forms identified in pancreatic extracts and released from the pancreas were proSS W92; proSS 65–76; an N-terminally extended form of proSS 65–76; and two larger forms comprising the proSS 20–36 sequence (but not the 1–13 sequence) with and without the proSS 65–76 sequence. hoSS 1–10, 1–32 and 65–92 (somatostatin 28) were not identified.

Nerve fibers in the rat posterior pituitary lobe contain prosomatostatin (1-64)

The neurohormone and neurotransmitter somatostatin arises from the processing of a larger precursor, prosomatostatin (proSS). An immunohistochemical investigation in the rat, using a well-characterized antiserum raised against a synthetic peptide identical to the 20-36 residues of the proSS molecule, revealed the presence of immunoreactive nerve fibers and nerve terminals in the median eminence, infundibulum, infundibular stalk and posterior pituitary lobe. The largest number of immunoreactive nerve fibers and nerve terminals was observed in apposition to the portal vessels, whereas a moderate number of proSS-immunoreactive fibers was identified in the infundibular stalk and in the proximal part of the posterior pituitary lobe. The proSS-immunoreactive nerves entered the posterior pituitary lobe from the infundibular and pituitary stalks and were followed to rostral and ventral aspects of the organ. In contrast, positive fibers were rarely identified in caudal and posterior parts. Extracts of rat posterior pituitaries subjected to gel chromatography and reversed-phase high-pressure liquid chromatography (HPLC) analysis showed the presence of a single proSS-immunoreactive molecule corresponding to the size of proSS(1-64). The functional significance of the proSS(1-64) in the hypothalamus and pituitary is at present unknown, but its location in the hypothalamo-hypophyseal system suggests that this end product of the processing of proSS is released into the portal and perhaps also the general circulation.