Liliana N. Berti-Mattera

Molecular Pharmacology of Human Vasopressin Receptors

Vasopressin (AVP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by activation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) AVP receptors and OT receptors (OTR). The cloning of the different members of the AVP/OT family of receptors now allows the extensive molecular pharmacological characterization of a single AVP/OT receptor subtype in stably transfected mammalian cell lines. The human V1-vascular (CHO-V1), V2-renal (CHO-V2), V3-pituitary (CHO-V3) and oxytocin (CHO-OT) receptors stably expressed in CHO cells display distinct binding profiles for 18 peptide and 5 nonpeptide AVP/OT analogs. Several peptide and nonpeptide compounds have a greater affinity for the V1R than AVP itself. V2R peptide agonists and antagonists tend to be non-selective ligands whereas nonpeptide V2R antagonists are potent and subtype-selective. None of the 22 AVP/OT analogs tested has a better affinity for the human V3R than AVP itself. Several peptide antagonists do not select well between V1R and OTR. These results underscore the need for developing specific and potent analogs interacting specifically with a given human AVP/OT receptor subtype.

Signal transduction pathways of the human V1-vascular, V2-renal, V3-pituitary vasopressin and oxytocin receptors.

Vasopressin (VP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by stimulation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) VP receptors and OT receptors (OTR). The recent cloning of the different members of the VP/OT family of receptors now allows the extensive characterization of the molecular determinants involved in ligand binding and signal transduction pathways coupled to a given VP/OT receptor subtype in stably transfected mammalian cell lines. In this article, we review the present knowledge of the signal transduction pathways coupled to the different VP/OT receptor subtypes and we present new observations derived from the study of each human VP or OT receptor subtype stably expressed in CHO cells.

Effects of p38 MAPK Inhibition on Early Stages of Diabetic Retinopathy and Sensory Nerve Function

Purpose. p38 mitogen-activated protein kinase (MAPK) is known to play a regulatory role in inflammatory processes in disease. Inflammation has been linked also to the development of diabetic retinopathy in rodents. This study was conducted to evaluate the effect of a p38 MAPK inhibitor on the development of early stages of diabetic retinopathy in rats. Methods. Streptozotocin-diabetic rats were assigned to two groups-treated with the p38 MAPK inhibitor PHA666859 (Pfizer, New York, NY) and untreated-and compared with age-matched nondiabetic control animals. Results. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal superoxide, nitric oxide (NO), cyclooxygenase (COX)-2, and leukostasis within retinal microvessels. All these abnormalities were significantly inhibited by the p38 MAPK inhibitor (25 mg/kgBW/d). At 10 months of diabetes, significant increases in the number of degenerate (acellular) capillaries and pericyte ghosts were measured in control diabetic rats versus those in nondiabetic control animals, and pharmacologic inhibition of p38 MAPK significantly inhibited all these abnormalities (all P < 0.05). This therapy also had beneficial effects outside the eye in diabetes, as evidenced by the inhibition of a diabetes-induced hypersensitivity of peripheral nerves to light touch (tactile allodynia). Conclusions. p38 MAPK plays an important role in diabetes-induced inflammation in the retina, and inhibition of p38 MAPK offers a novel therapeutic approach to inhibiting the development of early stages of diabetic retinopathy and other complications of diabetes.

Activin Induces Tactile Allodynia and Increases Calcitonin Gene-Related Peptide after Peripheral Inflammation

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide important in inflammatory pain that conveys pain information centrally and dilates blood vessels peripherally. Previous studies indicate that activin A increases CGRP-immunoreactive (IR) sensory neurons in vitro, and following wound, activin A protein increases in the skin and more neurons have detectable CGRP expression in the innervating dorsal root ganglion (DRG). These data suggest some adult sensory neurons respond to activin A or other target-derived factors with increased neuropeptide expression. This study was undertaken to test whether activin contributes to inflammatory pain and increased CGRP and to learn which neurons retained plasticity. After adjuvant-induced inflammation, activin mRNA, but not NGF or glial cell line-derived neurotrophic factor, increased in the skin. To examine which DRG neurons increased CGRP immunoreactivity, retrograde tracer-labeled cutaneous neurons were characterized after inflammation. The proportion and size of tracer-labeled DRG neurons with detectable CGRP increased after inflammation. One-third of CGRP-IR neurons that appear after inflammation also had isolectin B4 binding, suggesting that some mechanoreceptors became CGRP-IR. In contrast, the increased proportion of CGRP-IR neurons did not appear to come from RT97-IR neurons. To learn whether central projections were altered after inflammation, CGRP immunoreactivity in the protein kinase Cγ-IR lamina IIi was quantified and found to increase. Injection of activin A protein alone caused robust tactile allodynia and increased CGRP in the DRG. Together, these data support the hypothesis that inflammation and skin changes involving activin A cause some sensory neurons to increase CGRP expression and pain responses.

Transient expression of hypoxia-inducible factor-1 alpha and target genes in peripheral nerves from diabetic rats

Decreased blood flow is one of the earliest physiological changes observed after the onset of either clinical or experimental diabetes. The reduction in blood flow is believed to lead to nerve hypoxia, which in conjunction with other metabolic alterations and degenerative processes in different nerve compartments, results in the dysfunction known as diabetic neuropathy. The transcriptional regulator hypoxia-inducible factor-1 alpha (HIF-1alpha) accumulates rapidly under hypoxic conditions and modulates the expression of several target genes that protect tissues against ischemia and infarction. At present it is unclear whether diabetic nerve injury results from an abnormal response of HIF-1alpha and its protective target genes. In the present study we have analyzed the expression and activity of HIF-1alpha and its target genes in diabetic nerves as a first step to determine their possible contribution to the development or maintenance of diabetic neuropathy. We observed a transient increase in the expression of HIF-1alpha that peaked between 4 and 6 weeks and declined 8 weeks after induction of experimental diabetes in rats. The increase in HIF-1alpha in diabetic nerves coincided with a similarly transient increase in the expression of several HIF-1alpha target genes including vascular endothelial growth factor, lactate dehydrogenase and erythropoietin, which subsided 8-10 weeks after induction of diabetes. These results suggest that the transient activation of neurotrophic and angiogenic genes, as opposed to a more sustained effect in response to the chronic injury, may be responsible for the alterations in nerve function and regeneration that characterize the diabetic neuropathy.

Sulfasalazine Blocks the Development of Tactile Allodynia in Diabetic Rats

OBJECTIVE—Diabetic neuropathy is manifested either by loss of nociception (painless syndrome) or by mechanical hyperalgesia and tactile allodynia (pain in response to nonpainful stimuli). While therapies with vasodilators or neurotrophins reverse some functional and metabolic abnormalities in diabetic nerves, they only partially ameliorate neuropathic pain. The reported link between nociception and targets of the anti-inflammatory drug sulfasalazine prompted us to investigate its effect on neuropathic pain in diabetes. RESEARCH DESIGN AND METHODS—We examined the effects of sulfasalazine, salicylates, and the poly(ADP-ribose) polymerase-1 inhibitor PJ34 on altered nociception in streptozotocin-induced diabetic rats. We also evaluated the levels of sulfasalazine targets in sciatic nerves and dorsal root ganglia (DRG) of treated animals. Finally, we analyzed the development of tactile allodynia in diabetic mice lacking expression of the sulfasalazine target nuclear factor-κB (NF-κB) p50. RESULTS—Sulfasalazine completely blocked the development of tactile allodynia in diabetic rats, whereas relatively minor effects were observed with other salicylates and PJ34. Along with the behavioral findings, sciatic nerves and DRG from sulfasalazine-treated diabetic rats displayed a decrease in NF-κB p50 expression compared with untreated diabetic animals. Importantly, the absence of tactile allodynia in diabetic NF-κB p50−/− mice supported a role for NF-κB in diabetic neuropathy. Sulfasalazine treatment also increased inosine levels in sciatic nerves of diabetic rats. CONCLUSIONS—The complete inhibition of tactile allodynia in experimental diabetes by sulfasalazine may stem from its ability to regulate both NF-κB and inosine. Sulfasalazine might be useful in the treatment of nociceptive alterations in diabetic patients.

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen‐activated protein kinase and Ca2+‐independent phospholipase A2

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen‐activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet‐derived growth factor (PDGF), a known mitogen in these cells. Both endothelin‐1 (ET‐1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET‐1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET‐1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET‐1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca2+‐independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET‐1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET‐1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.

Reduced expression of endothelin B receptors and mechanical hyperalgesia in experimental chronic diabetes

Diabetic neuropathy is one of the major complications of diabetes mellitus. Small nerve fibers degenerate early in the disease, leading to symptoms ranging from hyperalgesia to loss of pain and temperature sensation. However, the cellular and molecular mechanisms responsible for abnormal pain perception in diabetes have not been identified. Both type-A and type-B endothelin receptors (ETAR and ETBR, respectively) are present in sensory nerves and appear to regulate neuropathic and inflammatory pain. In this study, we compared the expression of endothelin receptors and nociceptive responses in normal and experimentally diabetic rats. Diabetic animals exhibited both an increase in the withdrawal responses to high threshold stimuli (mechanical hyperalgesia) and to light touch stimuli (tactile allodynia). Immunohistochemical and Western blot analysis revealed that diabetic rats have significantly reduced expression of ETBR in sciatic nerves, while no changes were observed in dorsal root ganglia (DRG). In contrast, the expression of ETAR in either sciatic nerves or DRG of diabetic rats was not altered. Importantly, ETBR-deficient transgenic rats showed alterations in pain perception similar to those observed in diabetic rats. These results suggest that changes in the expression of ETBR in peripheral nerve may contribute to the development of mechanical hyperalgesia and tactile allodynia in chronic diabetes.

Hyperglycemia Triggers Abnormal Signaling and Proliferative Responses in Schwann Cells

Peripheral neuropathy is a serious diabetic complication. Delayed nerve regeneration in diabetic animal models suggests abnormalities in proliferation/differentiation of Schwann cells (SC). We recently reported that endothelins (ETs) regulate proliferation and phenotype in primary and immortalized SC (iSC). We now investigated changes in the effects of ETs on SC proliferation and signaling in nerve segments from streptozotocin-induced diabetic rats and in iSC exposed to high glucose. Cultured explants from diabetic rats displayed a delay in the time-course of [3H]-thymidine incorporation as well as enhanced sensitivity to endothelin-1 (ET-1) or insulin. iSC cultured in high (25 mM) glucose-containing media also exhibited higher [3H]-thymidine incorporation, along with an enhanced activation of p38 mitogen-activated protein kinase and phospholipase C in response to ET-1 or platelet-derived growth factor as compared to controls (5.5 mM glucose). These studies support an extra-vascular role of ETs in peripheral nerves and SC. The increased sensitivity to ET-1 in nerves and iSC exposed to high glucose may contribute to abnormal SC proliferation characterizing diabetic neuropathy.

Immortalized Schwann Cells Express Endothelin Receptors Coupled to Adenylyl Cyclase and Phospholipase C

Endothelins (ETs) are potent regulators of renal, cardiovascular and endocrine functions and act as neurotransmitters in the CNS. Here we report that immortalized Schwann cells express receptors for ETs and characterize some of the cellular events triggered by their activation. Specific binding of [125I]-ET-1 to Schwann cell membranes was inhibited by ET-1 and the ETB-selective agonists ET-3, sarafotoxin 6c and [A1a1,3,11,15]-ET-1 with IC50cor values ranging between 2 and 20 nM. No competition was observed with the ETA receptor-selective antagonist BQ123. Incubation of [3H]-inositol pre-labeled Schwann cells with ET-1, ET-3 or sarafotoxin 6c elicited a concentration-dependent increase in the release of IP1 that reached a plateau at approximately 100 nM. The efficacy of [Ala1,3,11,15]-ET-1 (a linear peptide analog of ET-1) was half of that corresponding to ET-1. These stimulatory effects were partially blocked by pre-incubation with pertussis toxin. When Schwann cells were incubated in the presence of 100 nM ET-1 or ET-3 there was a significant inhibition of basal and isoproterenol-stimulated cAMP levels. The inhibitory effects of sarafotoxin 6c and [Ala1,3,11,15]-ET-1 on isoproterenol-stimulated cAMP levels were similar to that observed with ET-1. Pre-incubation with pertussis toxin completely prevented this effect. These observations indicate that immortalized Schwann cells express receptors for ET peptides (predominantly ETB) coupled to modulation of phospholipase C and adenylyl cyclase activities. The actions of ETs on Schwann cells provide a novel example of the influence of vascular factors on nerve function.