Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.

The endogenous inhibitory role of the neuropeptide galanin in pain transmission and spinal cord excitability was demonstrated by the use of a high-affinity galanin receptor antagonist, M-35 [galanin-(1-13)-bradykinin-(2-9)-amide]. M-35, which displaced 12'I-labeled galanin from membranes of rat dorsal spinal cord with an IC50 of 0.3 nM, dose-dependently antagonized the effect of intrathecal galanin on the flexor reflex. M-35 potentiated the facilitation of the flexor reflex by conditioning stimulation of cutaneous unmyelinated afferents in rats with intact nerves and the potentiating effect of M-35 on the conditioning-stimulation-induced reflex facilitation of the cutaneous unmyelinated afferents was strongly enhanced after axotomy. These results demonstrate that endogenous galanin plays a tonic inhibitory role in the mediation of spinal cord excitability, and it is particularly noteworthy that this function of galanin is remarkably enhanced after peripheral nerve section.The neuropeptide galanin (1) consists of 29 amino acids, has been shown to occur in a relatively small population of dorsal root ganglion cells and spinal cord interneurons (2-4), and may be involved in sensory modulation. Although some reports indicated that intrathecal (i.t.) galanin has a hyperalgesic effect and i.t. galanin antibody produces analgesia (5), overwhelming evidence suggests that galanin primarily has an inhibitory effect in sensory transmission at the spinal level. Thus, i.t. galanin blocks the facilitatory effect of the flexor reflex produced by the conditioning stimulation (CS) of unmyelinated afferents and by i.t. applied excitatory neuropeptides, such as substance P and calcitonin gene-related peptide (6-8). Galanin also selectively depresses spinal nociceptive reflexes in adult and newborn rats with no or very limited effect on the monosynaptic reflex (6, 9, 10). Galanin (i.t.) has been reported to cause analgesia without motor deficits (11) and iontophresis ofgalanin hyperpolarizes dorsal horn neurons (12). Galanin also inhibits the peripheral consequences of antidromic activation of cutaneous unmyelinated (C) afferents, such as the plasma extravasation, mediated by the release of substance P (13,14).Peripheral axotomy has dramatic effects on peptidergic sensory afferents. Substance P and calcitonin gene-related peptide are downregulated whereas vasoactive intestinal peptide and galanin are upregulated (15)(16)(17)(18)(19)(20). We have recently demonstrated that the vasoactive intestinal peptide level is increased in the same sensory neurons that previously produced substance P (21) and that vasoactive intestinal peptide took over the role of tachykinins as excitatory mediators of nociceptive input (22). We have also shown that the depressive effect of galanin on the flexor reflex is enhanced after nerve section (23) and it antagonized the excitatory effect of vasoactive intestinal peptide, with which it coexists in axotomized sensory neurons (7). Based on these findings, we have hypothesized that one role for upr...

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The glanin-receptor Hlgand M40 [galanin-(1-12)-Pro3-(Ala-Leu)2-AIa amide] binds with high affinity to galanin-binding sites in hippocampal, hypothalamic, and spinal cord membranes and in membranes from Rin m5F rat insulinoma cells . Receptor autoradlographic studies show that M40 (1 "M) displaces galanin from binding sites in the hippocampus, hypothalamus, and spinal cord. In the brain, M40 acts as a potent galanin-receptor antagonist: M40, in doses comparable to that of galanin, antagonizes the stimulatory effects of glanin on feeding, and it blocks the galaninergic inhibition of the scopolamine-induced acetylcholne release in the ventral hippocampus in vivo. In contrast, M40 completely fails to antagonize both the galanin-mediated inhibition of the glucoseinduced insulin release in isolated mouse pancreatic islets and the inhibitory effects of galanin on the forskolin-stimulated accumulation of 3',5'-cAMP in Rin mSF cells; instead M40 is a weak agonist at the galanin receptors in these two systems. M40 acts as a weak antagonist of galanin in the spinal flexor reflex model. These results suggest that at least two subtypes of the glanin receptor may exist. Hypothalamic and hippocampal galanin receptors represent a putative central galaninreceptor subtpe (GL-1-receptor) that is blocked by M40. The pancreatic galanin receptor may represent another subtype (GL-2-receptor) that recognizes M40, but as a weak agonist. The galanin receptors in the spinal cord occupy an intermediate position between these two putative subtypes.Galanin is an important neuroendocrine peptide with multiple biological and pharmacological actions (1). It is a potent inhibitor of glucose-induced insulin release (2), it inhibits hippocampal acetylcholine release (3) induced by systemic administration of scopolamine (4), it impairs cognitive performance (5, 6), it stimulates feeding behavior upon hypothalamic or intracerebroventricular injection (7,8), it stimulates growth hormone secretion (9), and it has a biphasic effect on the spinal flexor reflex (10). Galanin hyperpolarizes noradrenergic cell bodies in the locus coeruleus (11) Accordingly, a galanin-receptor subtype has been suggested, which is composed of nervous tissue and pancreatic galanin receptors that recognize the N-terminal 1-to 15-aa or 1-to 16-aa fragment of galanin as high-affinity agonists, whereas another putative galanin-receptor subtype in smooth muscle requires both the N and C terminus of galanin for binding and biological action (25). On the basis of the differential affinity ofgalanin (3-29) and ofa galanin-receptor antagonist M15 (17), existence of an additional galaninreceptor subtype has been suggested in the rat anterior pituitary (26), which differs from other CNS galanin receptors. Finally, galanin-(1-15)-binding sites have been demonstrated in the dorsal hippocampus, neocortex, and neostriatum, areas that seem to lack galanin-(1-29)-binding sites (27).

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We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulininduced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.

The galanin N-terminal fragment [galanin-(1-16) The 29-amino-acid-long C-terminal amidated peptide galanin (GAL) (1) has been shown to be widely distributed in the central nervous system of mammals (2-6). GAL-like immunoreactivity has been localized, among other brain regions, in the septal area (4-8) where GAL coexists with acetylcholine in rat (7) and monkey (9) in a subpopulation of cholinergic cell bodies projecting to the hippocampus. and equilibrium binding studies with 1251I-labeled GAL (125I-GAL) have shown a high density of putative high-affinity GAL receptors in the ventral part of the hippocampus (13).In the ventral, but not in the dorsal, hippocampus GAL has been shown to inhibit, in a dose-dependent manner, the release of acetylcholine both in vitro and in vivo (12). A possible role for GAL as presynaptic modulator of the cholinergic function in the ventral hippocampus has been suggested by a recent electrophysiological study (14) and a behavioral study (15). Interactions between acetylcholine and GAL in the ventral hippocampus are not restricted to presynaptic sites but also involve actions that are considered postsynaptic (16) such as the GAL-mediated inhibition of the muscarinic agonist-stimulated breakdown of inositol phospholipids in the ventral hippocampus (17) and GAL inhibition of acetylcholine on a t-maze memory task in ventral forebrain lesioned rats (33).Previous studies carried out on intestinal smooth muscle showed the importance of the N-terminal portion of GAL for biological activity (18,19). Furthermore, N-terminal GAL fragments and analogs were able to mimic the effects of GAL on the pancreatic 83-cell line Rin m 5F, inhibiting forskolinstimulated cAMP production and insulin release (20).In this study, we focus our attention on the structureactivity relationship for the N terminus of GAL at receptors in the ventral hippocampus of the rat. Using equilibrium binding and autoradiographic techniques, we compare the synthetic N-terminal fragment GAL-(1-16) with rat GAL-(1-29) for its ability to displace 1251I-GAL from its receptors.GAL-(1-16) is also tested for its ability to inhibit the scopolamine-induced acetylcholine release in vivo and carbacholstimulated inositol phospholipid breakdown in a slice preparation from the rat ventral hippocampus. The importance of [Trp2] in the activity of the N-terminal fragment is examined.

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.