Biomedical subjects

P Ferrara

Publications and source records attributed to P Ferrara.

At least 91 records · Page 5Linked to original sources

PubMed1997

Interleukin-13 responsiveness and interleukin-13 receptor expression in non-Hodgkin's lymphoma and reactive lymph node B cells. Modulation by CD40 activation.

Interleukin-13 (IL-13) responsiveness was examined in lymph node B cells from patients with non-Hodgkin's lymphoma (NHL) and patients with benign reactive immune disorders. Proliferation assays showed that NHL B cells from 8 of 21 patients responded to IL-13 in the absence of a co-activation signal. IL-13-unresponsive NHL B cells from 9 of the 13 remaining patients were induced to respond to IL-13 upon antibody-triggered CD40 activation, as did reactive B cells. Binding experiments with radiolabeled IL-13 revealed that the constitutive expression of IL-13 receptors (IL-13R) was associated with IL-13 responsiveness in the absence of CD40 activation. In IL-13-unresponsive cells, IL-13R expression was induced after CD40 activation. This effect was enhanced by IL-10, which was able to potentiate the IL-13 response of CD40-activated cells. Furthermore, IL-13 was found to increase the viability of cultured NHL cells, but not that of non-malignant cells. These results suggest that IL-13, which behaves as a potent co-factor for normal lymph node B cell activation, might provide growth and/or survival advantages to NHL B cells.

B-Lymphocytes↗

PubMed1997

Congestive heart failure predicts the development of non-insulin-dependent diabetes mellitus in the elderly. The Osservatorio Geriatrico Regione Campania Group.

Congestive heart failure (CHF) is an insulin-resistant state which constitutes the main risk factor for the development of non-insulin-dependent diabetes mellitus (NIDDM). Our study investigated the predictive role of CHF on the development of NIDDM in 1,339 elderly subjects with a mean ( +/- SD) age of 74.2 +/- 6.4 years. CHF had a 9.5% prevalence, and 14.7% of the subjects had NIDDM. After stratification by age, subjects between 80 and 84 years had the highest prevalence of CHF and a total of 29.6% of CHF patients had NIDDM. In multiple logistic regression analysis, CHF was associated with NIDDM [odds ration (OR) = 2.0, 95% confidence interval (CI) - 1.6-2.5] independent of age, sex, family history of diabetes, body mass index, (BMI), waist/hip ratio, and diastolic blood pressure. When only untreated CHF patients were taken into account, the association between CHF and NIDDM was even stronger (OR = 4.0, 95% CI = 3.4-5.8). When untreated CHF patients were grouped into those with low (I and II) and high (III and IV) New York Heart Association (NYHA) classes, the association of CHF and NIDDM was stronger with the worsening of CHF. In a longitudinal study, CHF predicted NIDDM independently of age, sex, family history of diabetes, BMI, waist/hip ratio, systolic and diastolic blood pressure, and therapy for CHF (OR = 1.4, 95% CI = 1.1-1.8). CHF was associated with a higher prevalence of NIDDM and was a risk factor for its development. Elevated FFA concentrations may play a pivotal role.

Aged↗

PubMed1997

Interleukin-13 effects on activated monocytes lead to novel cytokine secretion profiles intermediate between those induced by interleukin-10 and by interferon-gamma.

We have examined in detail the activities of IL-13 on monokine production in vitro and compared its effects with those of IL-10 and IFN-gamma. IL-13 and IL-10 show qualitatively and quantitatively similar activities on cytokine production by monocytes when administered simultaneously with LPS i.e. inhibition of IL-1, IL-6 and TNF-alpha, up-regulation of IL1-ra. However when either LPS and IFN-gamma or fixed S. aureus Cowan (SAC) are used to activate monocytes, IL-10 is a much more potent inhibitor of TNF-alpha production than is IL-13. IL-10 is also an extremely potent inhibitor of IL-12 (p70) production when given with either SAC or LPS, while IL-13 has little effect. Indeed, IL-13 actually increases SAC-induced IL-12 production. When IL-13 is administered prior to the LPS stimulation, its modulation of cytokine production is drastically different. Production of IL-12, MCP-1, TNF-alpha and to a lesser extent IL-6 induced by LPS is now "primed", whereas that of IL-1, IL-8, and IL-10 is still inhibited. IL-10 does not show this "priming" effect, and is a dominant inhibitor of IL-13. The initial IL-13 priming effect is not however due to an inhibition of endogenous IL-10 production; nor is it due to inhibition of PGE2 production. The priming effect of IL-13 on IL-12 production is additive with that of IFN-gamma, and is partly independent of IFN-gamma. The earliest event in IL-13 priming so far noted is an increase in TNF-alpha mRNA production at 1-2 hours. IL-13 priming of IL-12 production can be completely abolished by anti-TNF-alpha antibodies suggesting that IL-13 may be priming via increased TNF-alpha expression, although merely substituting TNF-alpha for IL-13 does not reproduce the priming effect. IL-13 is a thus a more subtle immune regulator than IL-10 or IFN-gamma. When administered with LPS or SAC, it dampens the resulting inflammatory response, though in a more selective way than IL-10. In contrast, when it is added before an inflammatory signal, it primes an immunostimulatory monokine secretion profile resembling that of IFN-gamma, but without the proinflammatory IL-1 component. Early in response to an inflammatory stimulus, IL-13 may thus play an essentially anti-inflammatory role, switching to a primarily immunostimulatory role in the case of an ongoing infection.

Cytokines↗

PubMed1997

The related cytokines interleukin-13 and interleukin-4 are distinguished by differential production and differential effects on T lymphocytes.

We have compared the production of the related cytokines IL-13 and IL-4 by T lymphocytes, and the effects of the two cytokines on these cells. IL-13 and IL-4 production differ in a number of respects. IL-13 is produced at higher levels than IL-4 by activated T lymphocytes, and its accumulation in the culture medium can be more prolonged, corresponding partly to differential mRNA accumulation and partly to a preferential depletion of IL-4 from the culture medium. Certain inducing combinations such as PMA and anti-CD28, stimulate high levels of IL-13 and IL-13 mRNA, but little or no IL-4 or IL-4 mRNA. The ratio of IL-13 to IL-4, both at protein and mRNA levels, is higher in CD8+ lymphocyte than in CD4+ lymphocyte populations. Although after in vitro polarization of peripheral blood lymphocytes leading to type 1 and type 2 populations, IL-13 is made principally by cells of a type 2 phenotype, as is IL-4; it can also be produced by type 1 CD4+ and CD8+ T lymphocyte clones making large amounts of IFN-gamma and very little IL-4. IL-13 and IL-4 exert different effects on T lymphocyte functions. IL-13 does not significantly inhibit the IL-2-induced T lymphocyte production of IFN-gamma, RANTES, MIP-1 alpha or MIP-1 beta, nor that of perforin mRNA, as does IL-4. We have also been unable to demonstrate STAT6 activation by IL-13 on T lymphocytes purified in a number of ways, despite strong activation of STAT6 by IL-4 in these cells. This is contrary to some previous reports, but is consistent with the notion that the majority of T lymphocytes lack functional IL-13 receptors. A higher and more prolonged T lymphocyte production of IL-13 than that of IL-4 may thus be permissible because IL-13 does not inhibit T-cell functions. Conversely, sustained IL-13 production may be partly due to the absence of receptor-mediated depletion of this cytokine.

Base Sequence↗

PubMed1996

Cloning and characterization of a specific interleukin (IL)-13 binding protein structurally related to the IL-5 receptor alpha chain.

Interleukin-13 (IL-13) is a cytokine secreted by activated T lymphocytes that shares many, but not all, biological activities with IL-4. These overlapping activities are probably due to the existence of common receptor components. Two proteins have been described as constituents of the IL-4 receptor, a approximately 140-kDa glycoprotein (IL-4R) and the gamma chain (gammac) of the IL-2 receptor, but neither of these proteins binds IL-13. We have cloned a cDNA encoding an IL-13 binding protein (IL-13R) from the Caki-1 human renal carcinoma cell line. The cloned cDNA encodes a 380-amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The IL-13R shows homology with the IL-5 receptor, and to a lesser extent, with the prolactin receptor. COS-7 cells transfected with the IL-13R cDNA bind IL-13 with high affinity but do not bind IL-4. COS-7 cells co-transfected with the cloned IL-13R cDNA and IL-4R cDNA resulted in the reconstitution of a small number of receptors that recognized both IL-4 and IL-13. Reverse transcription-polymerase chain reaction analysis detected the receptor transcript only in cell lines known to bind IL-13.

Amino Acid Sequence↗

PubMed1996

Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of a levocabastine-sensitive neurotensin binding site.

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR-2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR-1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR-2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR-1, NTR-2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low-affinity binding sites in brain.

Amino Acid Sequence↗

PubMed1996

Structural features of the central cannabinoid CB1 receptor involved in the binding of the specific CB1 antagonist SR 141716A.

The antagonist SR 141716A has a high specificity for the central CB1 cannabinoid receptor and negligeable affinity for the peripheral CB2 receptor, making it an excellent tool for probing receptor structure-activity relationships. From binding experiments with mutated CB1 and with chimeric CB1/CB2 receptors we have begun to identify the domains of CB1 implicated in the recognition of SR 141716A. Receptors were transiently expressed in COS-3 cells, and their binding characteristics were studied with SR 141716A and with CP 55,940, an agonist recognized equally well by the two receptors. The region delineated by the fourth and fifth transmembrane helices of CB1 proved to be crucial for high affinity binding of SR 141716A. The CB1 and CB2 second extracellular loops, e2, were exchanged, modifications that had no effect on SR 141716A binding in the CB1 variant but that eliminated CP 55,940 binding in both mutants. The replacement of the conserved cysteine residues in e2 of CB2 by serine also eliminated CP 55,940 binding, but replacement of those in CB1 resulted in the sequestration of the mutated receptors in the cell cytoplasm. The e2 domain thus plays some role in CP 55,940 binding but none in SR 141716A recognition, binding of the latter clearly implicating residues in the adjoining transmembrane helices.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC.

Two new proteinases secreted by Cryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that the eapB and eapC genes contain three and two introns, respectively. The products of the eapB and eapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. The eapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of the eapC coding sequence in an endothiapepsin-deficient (EapA-) C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Amino Acid Sequence↗

PubMed1996

Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting.

In this study we used genetically manipulated strains in order to identify polypeptide spots of the protein map of Saccharomyces cerevisiae. Thirty-two novel polypeptide spots were identified using this strategy. They corresponded to the product of 23 different genes. We also explored the possibilities of using peptide-mass fingerprinting for the identification of proteins separated on our gels. According to this strategy, proteins contained in spots are digested with trypsin and the masses of generated peptides are determined by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). The peptide masses are then used to search a yeast protein database for proteins that match the experimental data. Application of this strategy to previously identified polypeptide spots gave evidence of the feasibility of this approach. We also report predictions on the identities of nine unknown spots using MALDI-MS.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1996

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

Animals↗

PubMed1996

Preconditioning does not prevent postischemic dysfunction in aging heart.

OBJECTIVES: This study was performed to investigate the effect of single or multiple brief periods of ischemia and the administration of exogenous norepinephrine before a more prolonged ischemic period and after reperfusion in adult and senescent isolated and perfused rat hearts. BACKGROUND: The mortality rate for coronary artery disease is greater in the elderly. Ischemic preconditioning has been proposed as an endogenous form of protection against ischemia-reperfusion injury. However, the role of preconditioning in aging heart is unknown. METHODS: We compared the protective effect of preconditioning transient ischemic and norepinephrine stimuli against 20 min of global normothermic ischemia and 40 min of reperfusion in isolated perfused hearts of adult (6 months old) and senescent (24 months old) rats. Norepinephrine release in coronary effluent was determined by high performance liquid chromatography. RESULTS: Final recovery of percent developed pressure was improved after single preconditioning transient ischemic and norepinephrine stimuli in adult hearts (87.7 +/- 9% and 82.3 +/- 8.7%) versus unconditioned control hearts (50.6 +/- 4.8%, p < 0.01 [mean +/-SD]). The effect of preconditioning on developed pressure recovery was not present in senescent hearts after transient ischemic stimulus (39.8 +/- 4.9% vs. 41.6 +/- 5.8%, p = NS) but was present after norepinephrine stimulus (74.3 +/- 10.5, p < 0.01). Norepinephrine release significantly increased after preconditioning transient ischemic stimulus in adult but not in senescent hearts (p < 0.01 vs. adult). Transient ischemic- and norepinephrine-induced preconditioning was blocked by alpha-adrenergic receptor antagonists in both adult and senescent hearts. Multiple transient ischemic stimuli were able to reduce postischemic dysfunction in adult but not in senescent hearts. CONCLUSIONS: Preconditioning transient ischemic stimulus significantly reduces postischemic dysfunction in adult but not in senescent hearts, whereas exogenous norepinephrine is able to mimic preconditioning in both adult and senescent hearts. Ischemic preconditioning induces an increase in norepinephrine release in adult but not in senescent hearts. Preconditioning induced by transient ischemic stimulus and norepinephrine was abolished by alpha-adrenergic receptor blockade in both adult and senescent hearts. Thus, our data demonstrate that preconditioning is absent in aging heart and is probably related to the reduction of norepinephrine release and alpha-adrenergic receptor stimulation in response to ischemic preconditioning.

Aging↗

PubMed1996

The role of aging on the control of contractile force by Na(+)-Ca2+ exchange in rat papillary muscle.

BACKGROUND: Sarcolemmal Na(+)-Ca2+ exchange system is believed to be fundamental to the control of cardiac contractility. However, the relation between Na(+)-Ca2+ exchange and the control of contractile force has not been studied in senescent myocardium. METHODS: The role of Na(+)-Ca2+ exchange in the regulation of the cardiac muscle's contractile force was studied in adult and senescent papillary muscles by simultaneously measuring intracellular sodium activity (aNai), action potential, and contractile force while varying extracellular concentration of sodium and/or calcium. RESULTS: Reduction of [Na+]o decreased aNai from 8.0 +/- 1.8 to 4.1 +/- 0.8 in adult (-3.9 mM) and from 8.7 +/- 1.9 to 4.7 +/- 0.9 in senescent (-4.0 mM) papillary muscles, while developed tension (DT) increased by 80.2% in adult and by 135.6% in senescent papillary muscles (p < .01 vs adult). During low [Ca2+]o and high [Na+]o, aNai and DT modifications were similar both in adult and senescent papillary muscles. During high [Ca2+]o, aNai decreased to a similar extent in both adult and senescent papillary muscles, while DT increased by 37.8% in adult and by 67.8% in senescent (p < .05 vs adult). Simultaneous reduction of [Na+]o and [Ca2+]o decreased aNai from 8.1 +/- 1.2 to 6.8 +/- 1.1 mM in adult (-1.3 mM), and from 8.4 +/- 1.0 to 7.2 +/- 1.0 mM in senescent (-1.2 mM) papillary muscles while DT decreased by 22.1% in adult and by only 12.0% in senescent (p < .01 vs adult) papillary muscles. Simultaneous increase of [Na+]o and [Ca2+]o similarly increased aNai in both adult senescent papillary muscles, but decreased DT by 28.5% in adult and by 11.7% in senescent (p < .01 vs adult). After [Na+]o modifications, the equilibration time for the ratio of external and internal sodium ion activities was slowed in senescent papillary muscles (i.e., in low [Na+]o solution the equilibration time was 4.6 +/- 0.9 min in adult and 6.3 +/- 1.2 min in senescent papillary muscles, p < .001). CONCLUSIONS: Similar changes of aNai during transmembrane Na+ and Ca2+ gradients modifications associated to changes in contractile force seem to demonstrate that Na(+)-Ca2+ exchange is probably not modified by the aging process. However, the slow equilibration time for the ratio of Na+ activities might reflect an age-related reduction of the Na(+)-K+ pump activity.

Action Potentials↗

PubMed1996

Interleukin-8 production by the human colon epithelial cell line HT-29: modulation by interleukin-13.

1. We have determined which cytokines induce and modulate the production of the chemokine interleukin-8 (IL-8) by the human colonic epithelial cell line HT-29. 2. Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-13 (IL-13), interleukin-10 (IL-10) or vehicle added alone or in combination. The production of IL-8 was determined by enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA expression by Northern blot analysis. 3. The production of IL-8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis. 4. HT-29 cells produced IL-8 following stimulation with IL-1 alpha or TNF-alpha in a time- and a concentration-dependent manner, while IFN-gamma, IL-10 and IL-13 did not induce IL-8 production by HT-29 cells. 5. IL-13 was found to up-regulate significantly (P < 0.01) the IL-1 alpha but not the TNF-alpha-induced IL-8 generation by HT-29 cells. In contrast, IL-10 had no effect on either IL-1 alpha or TNF-alpha-induced IL-8 production. 6. Experiments using cycloheximide demonstrated that this synergistic effect of IL-13 and IL-1 alpha on IL-8 secretion was not through de novo protein synthesis. Using actinomycin-D, we demonstrated that the IL-13 up-regulation was at the level of transcription rather than messenger RNA stability. 7. These findings suggest that colonic epithelial cells have a functional IL-13 receptor, which is coupled to an up-regulation of IL-1 alpha, but not TNF-alpha induced IL-8 generation.

Drug Synergism↗

PubMed1996

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.

Amino Acid Sequence↗

PubMed1996

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Amino Acid Sequence↗

PubMed1996

Characterization of two cloned human CB1 cannabinoid receptor isoforms.

We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.

Analgesics↗

PubMed1995

Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor.

The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently been characterized, structurally resembles opioid receptors and is negatively coupled with adenylate cyclase. ORL1 transcripts are particularly abundant in the central nervous system. Here we report the isolation, on the basis of its ability to inhibit the cyclase in a stable recombinant CHO(ORL1+) cell line, of a neuropeptide that resembles dynorphin A9 and whose amino acid sequence is Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln. The rat-brain cDNA encodes the peptide flanked by Lys-Arg proteolytic cleavage motifs. The synthetic heptadecapeptide potently inhibits adenylate cyclase in CHO(ORL1+) cells in culture and induces hyperalgesia when administered intracerebroventricularly to mice. Taken together, these data indicate that the newly discovered heptadecapeptide is an endogenous agonist of the ORL1 receptor and that it may be endowed with pro-nociceptive properties.

Amino Acid Sequence↗