Alpha1-adrenergic and dopaminergic receptors are involved in the anoretic effect of corticotropin-releasing factor in goldfish

This study investigates the noradrenergic and/or dopaminergic receptors subtypes involved in the anoretic action of CRF in goldfish. Agonists and antagonists of alpha1- and alpha2-adrenoceptors, and D1- and D2-dopaminergic receptors were intracerebroventricularly (i.c.v.) administered alone or in combination with CRF in the case of antagonists. Food intake and hypothalamic content of catecholamines and their metabolites were measured at 2 h postinjection. On one hand, alpha1-adrenergic receptor antagonist, but not alpha2, blocked the anoretic effect of CRF. Moreover, we found a blockade of CRF-induced anoretic action by pretreatment with specific D1- and D2-dopaminergic antagonists. On the other hand, the i.c.v. administration of CRF reduced hypothalamic norepinephrine (NE) and dopamine (DA) content, without modifications in their metabolism. Thus, our results suggest that the anoretic effect of CRF appears to be mediated by alpha1-adrenergic and dopaminergic receptors in fish. Second, the reduction in hypothalamic NE and DA synthesis could be due to a direct effect of CRF treatment and/or a secondary effect of food intake reduction.     

Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.     

The murine CYLN2 gene: genomic organization, chromosome localization, and comparison to the human gene that is located within the 7q11.23 Williams syndrome critical region

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2alpha, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a Gq-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor.     

Interaction between adrenaline and epidermal growth factor in the control of liver glycogenolysis in mouse

Epidermal growth factor (EGF) stimulates glycogenolysis in mouse liver, but the effect requires concentrations that are only achieved in plasma upon adrenergic stimulation of EGF release from submandibular salivary glands. Thus, we studied the interaction between adrenaline and EGF in liver glycogen metabolism, both in whole animals and in isolated hepatocytes. Adrenaline administered to anesthetized mice stimulated both the endocrine secretion of EGF from submandibular salivary glands and the degradation of glycogen in the liver. In sialoadenalectomized mice, adrenaline administration did not increase plasma EGF concentration. In these animals, the glycogenolytic response to adrenaline was enhanced. The sensitivity of hepatocytes to adrenaline was similar in cells from sialoadenalectomized and sham-operated mice. EGF, added to isolated hepatocytes, reduced the glycogenolytic effect of adrenaline (the maximal effect but not the ED50). Adrenaline stimulated glycogen degradation through both an alpha1-adrenergic mediated Ca2+ increase and a beta-adrenergic-mediated cAMP increase. EGF did not interfere with the rise of cytosolic Ca2+ but decreased the cAMP signal. EGF did not decrease the glycogenolytic effect of phenylephrine or VP (which increased cytosolic Ca2+ but not cAMP), but EGF decreased both the glycogenolytic effect and the cAMP signal generated by glucagon or forskolin. EGF did not interfere with the glycogenolytic effect of CPT-cAMP or bt2-cAMP. The effect of EGF on cAMP was blocked by 3-isobutyl-1-methylxanthine. These results demonstrate that the effect of EGF on the glycogenolytic action of adrenaline involves interference with the generation of the cAMP signal. We suggest that EGF induces such an effect through the activation of a phosphodiesterase.     

Endogenous norepinephrine regulates tumor necrosis factor-alpha production from macrophages in vitro

Evidence for the extraneuronal accumulation of norepinephrine has been demonstrated to occur in macrophage (M phi), yet the physiologic role of this system remains undefined. We have assessed the response of murine peritoneal M phi to adrenergic antagonists. We have also defined a physiologic role of a M phi-associated pool of the nonspecific adrenergic agonist norepinephrine. We investigated the constitutive involvement of alpha-adrenergic and beta-adrenergic receptors in LPS-induced TNF-alpha production. CFA-elicited M phis were incubated with LPS (1 microgram/ml) in the presence or absence of adrenergic agonists and/or antagonists. Although stimulation of alpha-adrenergic receptors increased TNF production and gene expression, beta-adrenergic receptors decreased it. Interestingly, when adrenergic antagonists along with LPS alone were added to M phi, they generated the response opposite to that produced by their suitable agonist, suggesting a role for endogenous norepinephrine in M phi. Thus, although alpha 2-adrenergic antagonists attenuated TNF production, beta-adrenergic antagonists augmented TNF expression in a concentration-dependent manner. Norepinephrine and epinephrine were found in M phi as determined by HPLC and LPS stimulation induced a significant decrease in their content. M phis were also incubated with LPS or medium only, washed, and then challenged 12 h later with LPS. When given a second LPS stimulation, M phis were found to have an increased response to alpha 2-adrenergic agonists and decreased response to alpha 2-adrenergic antagonists. Therefore, M phi-associated norepinephrine appears to regulate LPS-induced TNF production in an autocrine fashion.     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.     

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.     

Dose-response relationship between total cadmium intake and beta 2-microglobulinuria using logistic regression analysis

Noradrenaline stimulates not only Ca2+ mobilization but also cAMP formation through activation of alpha 1-adrenoceptors in hepatocytes from mature male rats. We examined which subtype(s) of alpha 1-adrenoceptor mediate these signal transduction mechanisms. Treatment of hepatocytes with chloroethylclonidine produced a dose-dependent inhibition of noradrenaline-induced Ca2+ mobilization, involving both transient and sustained components. Chloroethylclonidine also blocked noradrenaline-induced cAMP accumulation. It was observed that prazosin was much more potent than WB4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane) in antagonizing noradrenaline-induced Ca2+ mobilization. The same potency order was found in cAMP formation studies. Pretreatment of rats with pertussis toxin did not affect alpha 1-adrenergic responsiveness. Incubations of hepatocytes with tumor-promoting phorbol esters eliminated both Ca2+ mobilization and cAMP accumulation caused by noradrenaline. Our data suggest that in hepatocytes from mature male rats, single alpha 1B-adrenoceptors are linked to cAMP formation as well as Ca2+ mobilization.     

Role of endoscopic injection therapy in the treatment of bleeding peptic ulcer

Transforming growth factor beta (TGF beta) was examined regarding its regulation of the mitogen EGF. A431 human epidermoid carcinoma cells were treated with TGF beta and epidermal growth factor (EGF) (10 ng/ml each) to determine if TGF beta modulates EGF-induced Ca2+ signaling and c-Fos oncoprotein levels. Changes in [Ca2+]i were determined by digital imaging analysis or photon counting. In HBSS + Ca2+ (1.37 mM), EGF treatment resulted in a transient increase in [Ca2+]i from 75 to 150 nM, which lasted approximately 3.5 min and re-equilibrated to 90 nM. In nominally Ca(2+)-free (2-5 muM) HBSS, EGF caused a [Ca2+]i elevation that peaked at 140 nM and returned to baseline. TGF beta in HBSS + Ca2+ did not elicit a [Ca2+]i increase, although affinity labeling revealed types I, II, and III TGF beta receptors. TGF beta added simultaneously with EGF in HBSS + Ca2+ caused a gradual rise in [Ca2+]i from 50 to 100 nM over 16 min. Pretreatment with TGF beta (3 h; 10 ng/ml) abolished the EGF-induced [Ca2+]i elevation. EGF or TGF beta treatments increased c-Fos immunoreactivity by around 1 h. In summary, EGF elevated [Ca2+]i in the presence or absence of [Ca2+]e, resulting in high [Ca2+]n, associated with tyrosine and threonine phosphorylation, and increased c-Fos oncoprotein immunoreactivity. TGF beta did not increase [Ca2+]i but did increase c-Fos; TGF beta + EGF added simultaneously altered the EGF-induced [Ca2+]i elevation, and TGF beta pretreatment eliminated EGF-induced [Ca2+]i elevation. This suggests that TGF beta can regulate EGF in A431 cells and that increased c-Fos may not be mediated by Ca2+.     

Brain alpha 2-adrenergic receptor binding during incomplete cerebral ischemia in the rat

alpha 2-Adrenergic agonists decrease sympathetic activity and improve outcome from brain ischemia. We evaluated whether changes in alpha 2-adrenergic receptor binding activity may be important in the sympathetic depressant and cerebral protective effects of halothane (1.1% inspired) or isoflurane (1.4% inspired) compared to fentanyl/nitrous oxide (N2O) anesthesia. Brain alpha 2-adrenergic receptor binding was measured using [3H]-clonidine in each of four treatment conditions: 1, unanesthetized; 2, anesthetized (fentanyl/N2O, halothane, or isoflurane): 3, anesthetized with ischemia; 4, after 4 h recovery from ischemia. Ischemia was produced by right carotid artery ligation combined with hemorrhagic hypotension to 30 mm Hg for 30 min. Both halothane and isoflurane decreased alpha 2-adrenergic receptor density 20% compared to unanesthetized values (P < 0.01). This decrease was attenuated in ischemic tissue. There were no consistent changes in receptor affinity. These results suggest that inhaled anesthetics decrease the number of alpha 2-adrenergic receptors. This decrease appears to be unrelated to plasma catecholamine concentrations but may be influenced by the degree of ischemia.     

Insulin stimulates sequestration of beta-adrenergic receptors and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the beta2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of beta2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing beta2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of beta-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the beta-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with beta-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to beta2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-agonist, or both. The Tyr-350 --> Phe mutant form of the beta2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of beta2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the beta-adrenergic receptor, via a new mechanism, i.e. internalization.     

Rilmenidine elevates cytosolic free calcium concentration in suspended cerebral astrocytes

Rilmenidine, a ligand for imidazoline and alpha2-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca2+ concentration ([Ca2+]i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca2+]i, consisting of a transient increase (1-100 microM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1-10 mM rilmenidine). A similar elevation in [Ca2+]i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca2+-free medium, indicating that rilmenidine evokes release of Ca2+ from intracellular stores. However, the sustained elevation of Ca2+ was completely dependent on extracellular Ca2+, consistent with rilmenidine activating Ca2+ influx. Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca2+ influx pathway. The alpha2-adrenergic antagonist rauwolscine attenuated the increase in [Ca2+]i induced by clonidine (a selective alpha2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca2+ release from intracellular stores and Ca2+ influx by a mechanism independent of alpha2-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.     

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

OBJECTIVE: Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN: Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS: Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION: These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.     

Invasion depth diagnosis of depressed type early colorectal cancers by combined use of videoendoscopy and chromoendoscopy

The activation of rat mesocortical dopaminergic (DA) neurons evoked by the electrical stimulation of the ventral tegmental area (VTA) induces a marked inhibition of the spontaneous activity of prefrontocortical cells. In the present study, it was first shown that systemic administration of either clozapine (a mixed antagonist of D1, D2, and alpha1-adrenergic receptors) (3-5 mg/kg, i.v.), prazosin (an alpha1-adrenergic antagonist) (0.2 mg/kg, i.v.), or sulpiride (a D2 antagonist) (30 mg/kg, i.v.), but not SCH 23390 (a D1 antagonist) (0.2 mg/kg, i.v.), reversed this cortical inhibition. Second, it was found that following the systemic administration of prazosin, the VTA-induced cortical inhibition reappeared when either SCH 23390 or sulpiride was applied by iontophoresis into the prefrontal cortex. Third, it was seen that, whereas haloperidol (0.2 mg/kg, i.v.), a D2 antagonist which also blocks alpha1-adrenergic receptors, failed to reverse the VTA-induced inhibition, the systemic administration of haloperidol plus SCH 23390 (0.2 mg/kg, i.v.) blocked this inhibition. Finally, it was verified that the cortical inhibitions obtained following treatments with either "prazosin plus sulpiride" or "prazosin plus SCH 23390" were blocked by a superimposed administration of either SCH 23390 or sulpiride, respectively. These data indicate that complex interactions between cortical D2, D1, and alpha1-adrenergic receptors are involved in the regulation of the activity of prefrontocortical cells innervated by the VTA neurons. They confirm that the physiological stimulation of cortical alpha1-adrenergic receptors hampers the functional activity of cortical D1 receptors and suggest that the stimulations of cortical D1 and D2 receptors exert mutual inhibition on each other's transmission.     

Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.     

Effects of epidermal growth factor and insulin on migration and proliferation of primary cultured rabbit gastric epithelial cells

We assessed the influence of epidermal growth factor (EGF) and insulin on gastric epithelial restoration in vitro. Rabbit gastric epithelial cells were cultured and formed a complete monolayer cell sheet in 2 days. We created a wound (1.8 +/- 0.05 mm2) by denuding an area of cells, and EGF (0.1-30 ng/ml) and/or insulin (1 nM-1 microM) was added. The restoration process, which included cell migration and proliferation, was monitored by measuring the cell-free area every 12 h for 2 days. Proliferating cells were detected by sequential staining with bromodeoxyuridine (BrdU). Control cells showed complete repair in 36-48 h and restoration was accelerated dose-dependently by EGF or insulin. EGF plus insulin further accelerated restoration, which was then completed in 12-24 h. EGF and/or insulin increased the number of BrdU- positive cells. The results indicated that EGF and insulin additively accelerated gastric epithelial wound repair by stimulating both the migration and the proliferation of gastric epithelial cells (particularly the former).     

The delta-opioid receptor in SK-N-BE human neuroblastoma cell line undergoes heterologous desensitization

The human neuroblastoma cell line SK-N-BE expresses delta-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 nM etorphine leads to an almost complete desensitization (8.2 +/- 5.9 vs. 45.8 +/- 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, alpha 2-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 microM 2-bromo-alpha-ergocryptine or 1 microM arterenol resulted in a marked desensitization of D2-dopaminergic and alpha 2-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the delta-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-brorno-alpha-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the alpha 2-adrenergic receptor response, whereas pretreatment with 1 microM arterenol partly desensitized the delta-opioid receptor response. It is concluded that the delta-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterolgous desensitization, and it is suggested that delta-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G12 protein, whereas alpha 2-adrenergic receptor could be coupled to the enzyme via two G proteins, G12 and another member of the G1/G0 family.