Inflammatory disease in HLA-B27 transgenic rats.

UNLABELLED: A spontaneous inflammatory disease in rats transgenic for HLA-B27 resembles the B27-associated human spondyloarthropathies. Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells. Control rats with HLA-B7 remain healthy. Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease. Rats with a mutant B27 with a Cys67-->Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis. A similar phenotype is seen in B27 rats co-expressing a viral peptide that binds B27. Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression. Colitis is associated with high interferon-gamma, arthritis with high interleukin-6. Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective. CONCLUSIONS: The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67. Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity. Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility. The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.

Regulation of guanine nucleotide turnover on Gi/Go by agonist-stimulated and spontaneously active muscarinic receptors in cardiac membranes.

Muscarinic receptor regulation of guanine nucleotide turnover on Gi/Go proteins in ventricular sarcolemma was investigated. In the absence of a muscarinic receptor (MR) agonist, GTP bound to background sites with a Kapp value of 60 nM and a Bmax of 50 pmol/mg. The addition of the MR agonist, carbachol, further increased GTP binding by 50 pmol/mg to sites with the same Kapp value of 60 nM. Pertussis toxin treatment reduced GTP binding to carbachol-regulated and background binding sites, thus identifying both sites as Gi/Go. The identity of the carbachol-regulated GTP binding sites was further confirmed by demonstrating that carbachol stimulated GTP binding and inhibited adenylyl cyclase with an EC50 value of 200 nM. Background and carbachol-regulated guanine nucleotide binding sites bound GDP with a Kapp value of 150 nM. However, maximal background GDP binding was 50 pmol/mg, whereas maximal carbachol-regulated GDP binding was only 12-15 pmol/mg. In sarcolemma preloaded with [3H]GDP, carbachol-regulated [3H]GDP release was strictly dependent on the presence of guanine nucleotides. The Kapp values for GTP and GDP to support carbachol-regulated [3H]GDP release were 60 nM and 150 nM, respectively. Guanosine 5'-O-(3-thiotriphosphate) (GDPbetaS) facilitated carbachol-regulated [3H]GDP release with a Kapp value of 2 microM. However, GTP was two times more efficacious than GDP or GDPbetaS in facilitating carbachol-regulated [3H]GDP release. Mn2+ also stimulated [3H]GDP release from carbachol-regulated sites by a mechanism not requiring guanine nucleotides. These studies indicate that two pools of muscarinic receptors, carbachol regulated and spontaneously active, regulate guanine nucleotide turnover on pertussis toxin sensitive Gi/Go. These studies further suggest that guanine nucleotide binding provides the signal to stimulate GDP release from receptor activated Gi/Go proteins. A quaternary mechanism involving G-protein interactions may be necessary to promote guanine nucleotide exchange on Gi/Go.

The specificity of peptides bound to human histocompatibility leukocyte antigen (HLA)-B27 influences the prevalence of arthritis in HLA-B27 transgenic rats.

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27(+)NP1(+) rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced approximately 90% of the 3H-Arg-labeled endogenous peptide fraction in B27(+)NP1(+) spleen cells. Male B27(+)NP1(+) rats had a significantly reduced prevalence of arthritis, compared with B27(+)NP- males or B27(+) males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER.

Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease.

Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cima and cimb, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cima B27 transgenic rats lysed male B27 cimb/b targets significantly less well than cima/a or cima/b targets. Addition of exogenous H-Y peptides to male B27 cimb/b targets increased susceptibility to lysis to the level of cima/a targets. Male B27 cimb/b cells were less efficient than cima/a cells in competitively inhibiting CTL lysis of female B27 cima/a targets sensitized with exogenous H-Y peptides. 3H-Labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cimb and three cima RT1 haplotypes showed that the cimb peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cima/b or cimb/b. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism.

Modification of cardiac membrane adenylate cyclase activity and Gs alpha by NAD and endogenous ADP-ribosyltransferase.

The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate Gs (i.e. 5 mM AlF4-, 10 microM GTP gamma S, 10 microM GppNHp or isoproterenol plus 2 nM GTP gamma S). Furthermore, the EC50 of isoproterenol to stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 microM GTP gamma S or isoproterenol plus 2 nM GTP gamma S produced a selective increase in the radiolabeling of a single 45-kDa protein which was identified as Gs alpha by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from Gs alpha prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on Gs alpha was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of Gs alpha and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of Gs alpha was not removed under hypotonic or hypertonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess an ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated Gs alpha.

Regulation of polyphosphoinositide synthesis in cardiac membranes.

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.

Contribution of the vasopressin V1 receptor to its hydrosmotic response.

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water-soluble inositol phosphates when treated with 10(-8) M vasopressin (AVP), but not with (1-deamino-8-D-arginine)vasopressin (dDAVP), a V2-agonist. The increase in inositol phosphates was blocked by the V1-antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1-coupled phosphoinositide breakdown. The V1-antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1-antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1-receptor cascade in the water flow response. Mezerein (MZ), a non-phorbol activator of protein kinase C (PKC) increased osmotic water flow when added to the mucosal surface. The response was less in magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1-response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP-mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1-receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1 receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability that occurs at the apical membrane.

Muscarinic receptor stimulated phosphoinositide turnover in cardiac atrial tissue.

The properties of muscarinic agonist stimulated phosphoinositide turnover in canine atrial slices were investigated. In slices prelabeled with 32Pi, carbachol stimulated a 20-30% decrease of 32P-labeled phosphatidylinositol 4'-phosphate (PIP) and phosphatidylinositol 4',5'-bisphosphate (PIP2) content within 10-15 sec. This was followed by a resynthesis of these lipids to control levels after 30 sec. Carbachol-stimulated PIP and PIP2 turnover was followed by a relatively slower increase in 32P incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) which was maximal after 5-10 min. Carbachol increased 32P-labeling of PA and PI in most regions of right and left atria with equal effectiveness. Muscarinic receptor stimulated increases in PA and PI labeling showed high specificity for certain muscarinic agonists and, unlike most tissues, this muscarinic receptor mediated phospholipid effect was dependent on extracellular calcium. Carbachol did not increase 32P incorporation into PA and PI if Mn2+, Co2+, Mg2+, or La3+ was substituted for extracellular Ca2+. Unlike other muscarinic agonists, acetylcholine increased 32P incorporation into phosphatidylcholine in addition to PA and PI. Low concentrations of calcium channel blockers, verapamil, nifedipine or diltiazem, did not block carbachol-stimulated changes in PA and PI labeling in the presence of Ca2+; however, higher concentrations (greater than or equal to 10 microM) of verapamil increased PA and PI labeling. Ouabain enhanced carbachol-stimulated 32P incorporation into PA but attenuated incorporation into PI. The mechanisms associated with the actions of these agents on phospholipid metabolism and their possible physiological significance are discussed.

Effects of antiglaucoma drugs on [32P]orthophosphate incorporation into phospholipids of cat iris and ciliary process.

The effects of antiglaucoma drugs on [32P]-orthophosphate incorporation into phospholipids of iris and ciliary process were investigated. Both iris and ciliary process rapidly incorporated 32Pi into the major phospholipids, with the acidic phosphoinositides demonstrating a greater labelling than phosphatidylcholine, indicating a greater turnover. The muscarinic agonists, carbachol and pilocarpine, stimulated 32Pi-labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in both iris and ciliary process. These effects were blocked by atropine, suggesting that the response was mediated through muscarinic receptors. The beta blocking ocular hypotensive drugs, propranolol, timolol and atenolol, produced varying effects on 32P incorporation into phospholipids of iris and ciliary process. Propranolol stimulated 32Pi-labelling into phosphatidylinositol 4', 5' bisphosphate (PIP2), phosphatidylinositol 4' phosphate (PIP), PI and PA. Timolol decreased 32Pi-incorporation into PIP2 and PI, whereas atenolol, a selective beta 1 antagonist, had no significant effect on 32Pi-labelling of phospholipids. The above findings on propranolol agree with previous observations which demonstrated that propranolol redirects glycerolipid metabolism through multiple effects on the enzymes in phospholipid biosynthesis, particularly in stimulating phosphatidylinositol kinases. The results with timolol suggest that this drug may decrease phosphoinositide hydrolysis. The effects of these ocular hypotensive, non-selective beta blocking drugs on phospholipid turnover may ultimately limit the accumulation of breakdown products which could serve as cellular messengers.