Effect of In Vivo Modulation of Membrane Docosahexaenoic Acid Levels on the Dopamine-Dependent Adenylate Cyclase Activity in the Rat Retina

Abstract: We have studied the effect of a dietary deprivation of n-3 fatty acids on the activity of the dopamine (DA)-de-pendent adenylate cyclase in the rat retina. Experiments were conducted in 6-month-old rats raised on semipurified diets containing either safflower oil (n-3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n-3)] in retinal phospholipids were significantly decreased in n-3 deficient rats (35–42% of control levels). This was compensated by a rise in 22:5 (n-6), the total content of poly-unsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark-adapted or light-exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light-dependent fashion. The activation was lower in rats exposed to light than in dark-adapted animals, suggesting a down-regulation of the DI DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light-exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide- and DA-dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n-3 to n-6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.     

Presence of Corticotropin-Releasing Factor-Stimulated Adenylate Cyclase Activity in Rat Retina

Corticotropin-releasing factor (CRF) stimulates rat retinal adenylate cyclase activity in a concentration-dependent manner. The half-maximal effect is obtained at 50 nM CRF and the maximal stimulation corresponds to approximately 90% increase of basal enzyme activity. The CRF effect is counteracted by the CRF antagonist alpha-helical CRF 9-41 with a Ki value of 40 nM. Other CRF-like peptides such as sauvagine and urotensin I are as effective as CRF with a rank order of potency of urotensin I greater than or equal to sauvagine greater than CRF. The sauvagine and urotensin I effects are not additive with that elicited by CRF. Moreover, the CRF stimulation is not additive with the increase of enzyme activity produced by vasoactive intestinal peptide or dopamine. The CRF effect is independent of the concentration of free Ca2+, is optimal at 5-10 mM MgCl2, and requires GTP. The results indicate that rat retinal adenylate cyclase is modulated by CRF via a receptor-mediated mechanism.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Postmortem Stability of Dopamine-Sensitive Adenylate Cyclase, Guanylate Cyclase, ATPase, and GTPase in Rat Striatum

The stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+-dependent guanylate cyclase activities showed no significant change over this period. Mn2+-dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine-stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine-sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.     

Properties of Detergent-Dispersed Adenylate Cyclase from Cerebral Cortex. Presence of an Inhibitor Protein

Abstract: Adenylate cyclase was solubilized from washed paniculate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca²⁺ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca²⁺ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca²⁺-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca²⁺-stimulated adenylate cyclase in the Ca²⁺-sensitive fraction. The inhibitor was inactivated by heating at 60°C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Reconstitution of Calmodulin-Sensitive Adenylate Cyclase from Bovine Brain with Phosphatidylcholine Liposomes

A partially purified calmodulin (CaM)-sensitive adenylate cyclase from bovine cerebral cortex was reconstituted with a series of phosphatidylcholine liposomes having variable fatty acid composition. The enzyme was successfully associated with dimyristoyl, dipalmitoyl, distearoyl, and dioleoylphosphatidylcholine liposomes. The specific activity of the enzyme in the various liposomes varied over a 4.6-fold range indicating some degree of specificity for fatty acid composition. The adenylate cyclase-liposome preparation retained sensitivity to both CaM and 5'-guanylylimidodiphosphate (GppNHp). Arrhenius plots of enzyme activity in the four different liposome preparations all exhibited a pronounced discontinuity at 30 degrees C +/- 2, even though the bulk-phase thermal transition points for the liposomes varied from -20 to 54 degrees C. Fluorescence anisotropy studies of reconstituted liposome systems illustrated that incorporation of protein did not alter the normal-phase transition point of these lipids. Since Arrhenius plots of the enzyme in Lubrol PX, prior to reconstitution with lipids, were strictly linear, it is concluded that the breaks at 30 degrees C may be the effect of a local enzyme-phospholipid environment. It appears that this adenylate cyclase is not particularly sensitive to phase transitions of the bulk lipid phase. The phospholipid reconstituted enzyme system appears suitable for examination of the influence of lipids on the CaM-sensitive adenylate cyclase.     

Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat

Abstract: Adenylate cyclase activity in cell-free homogenates of the rat superior cervical ganglion (SCG) was assayed under a variety of experimental conditions. Adenylate cyclase activity was decreased by approximately one-half when 1 m M EGTA was included in the homogenization buffer and assay mixture, indicating the presence of a Ca²⁺-sensitive adenylate cyclase in the ganglion. In the presence of EGTA, basal adenylate cyclase activity in homogenates of the SCG was 12.9 ± 0.6 pmol cyclic AMP/ganglion/10 min. Enzyme activity was stimulated three- to fourfold by 10 m M NaF or 10 m M MnCl₂, Both GTP and its nonhydrolyzable analog guanylylimidodiphosphate (GppNHp) stimulated adenylate cyclase in a concentration-dependent manner over the range of 0.1–10.0 μ M . Stimulation by GppNHp was five to six times greater than that produced by GTP at all concentrations tested. Decentralization of the ganglion had no effect on basal or stimulated adenylate cyclase activity. Receptor-linked stimulation of adenylate cyclase was not obtained with any of the following: isoproterenol, epi-nephrine, histamine, dopamine, prostaglandin E₂, or va-soactive intestinal peptide. Thus the receptor-linked regulation of adenylate cyclase activity appears to be lost in homogenates of the ganglion.     

Docosahexaenoic Acid Is a Major n-3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µ M ) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.     

Ethanol''s Effects on Cortical Adenylate Cyclase Activity

The effects of ethanol on beta-adrenergic receptor-coupled adenylate cyclase (AC) of mouse cerebral cortex were examined. The addition of ethanol (20-500 mM) to incubation mixtures containing cortical membranes demonstrated that ethanol could increase AC activity and potentiate the stimulatory effects of guanylyl-imidodiphosphate [Gpp(NH)p] on AC activity. Ethanol increased the rate of activation of AC by guanine nucleotides and concomitantly decreased the EC50 for magnesium required to achieve maximal stimulation of cortical AC. The EC50 values for Gpp(NH)p and isoproterenol stimulation of AC activity were also altered by ethanol. Ethanol was capable of stimulating AC extracted by use of digitonin. The AC activity in the digitonin extract was no longer sensitive to the addition of Gpp(NH)p or NaF, but was still stimulated by ethanol. We propose multiple sites of action for ethanol in stimulating cortical AC activity. These sites include actions at the beta-adrenergic receptor, at the G/F coupling proteins, and at the catalytic unit of cortical AC. Comparison of ethanol's actions on cortical beta receptor coupled AC activity with prior reported actions of ethanol on striatal dopamine (DA)-sensitive AC indicated differential sensitivities of these two AC systems to ethanol. These differences may be determined by specific coupling characteristics of the striatal and cortical AC systems or by differences in the plasma membranes in which striatal and cortical AC systems are located.     

Quantitation of Adenylate Cyclase ofBordetella pertussisby Enzyme Linked Immunosorbent Assay

Bordetella pertussisproduces extracytoplasmic adenylate cyclase toxin (AC) which has received considerable attention as a potential vaccine candidate. Great interest from laboratories involved in production, purification and quality control of acellular pertussis vaccine is focused on finding an appropriate technique for rapid and accurate quantitation of AC antigen. In this paper a competitive ELISA is proposed. A polystyrene microplate coated with purified AC was incubated with the sample to be tested plus anti-AC serum. The bound anti-AC antibodies were measured by sequential reaction with alkaline phosphatase-labelled anti-mouse IgG and p-nitrophenylphosphate. This method showed high specificity, with the 50% inhibition corresponding to 4 μg/ml of AC. It also proved to be useful to assess the presence of AC in culture supernatants, with high reproducibility.     

Multiple Defects in the Activity of Adenylate Cyclase from the Drosophila Memory Mutant Rutabaga

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.     

Structure of Somatostatin Isolated from Bovine Retina

Abstract: Somatostatin-like immunoreactivity (SLI) from bovine retina was purified and its structure determined. Retinal tissue (1868 g) extracted with 3% acetic acid yielded 18.6 nmol SLI. This peptide was purified by chromatography on an affinity column made with anti-somatostatin antiserum, a reverse-phase C-18 HPLC column, and three sequential applications on a reverse-phase phenyl HPLC column. The peptide was purified 103,000-fold from the initial extract with an overall yield of 14.4%. Amino acid sequence determination by an automatic Edman degradation technique revealed the sequence to be as follows: Ser - Ala - Asn - Ser - Asn - Pro - Ala - Met - Ala - Pro - Arg - Glu - Arg - Lys - Ala - Gly - (Cys) - Lys - Asn - Phe - Phe - Trp - Lys - Thr - (Phe, Thr, Ser, Cys). The apparent identity of this peptide with somatostatin octacosapeptide (S28) purified from other mammalian tissue indicates the phylogenetic conservation of its structure and facilitates the use of the retina as a model system for studying the neurotransmitter function of somatostatin.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Interactions Between Vasoactive Intestinal Peptide and Dopamine in the Rabbit Retina: Stimulation of a Common Adenylate Cyclase

Although 3,4-dihydroxyphenylethylamine (dopamine, DA) and vasoactive intestinal peptide (VIP) have been reported to stimulate adenylate cyclase activity in the rabbit retina, possible interactions between VIP-sensitive and DA-sensitive adenylate cyclase systems have not been previously investigated. To elucidate the interactions between these two putative transmitter-stimulated cyclase systems, the effects of VIP, DA, and VIP + DA on the conversion of [alpha-32P]ATP to [32P]cyclic AMP in rabbit retinal homogenates were measured. VIP stimulated adenylate cyclase activity in a biphasic manner, suggesting that two classes of VIP receptors may be involved in the induction of cyclic AMP formation. DA was less potent than VIP, and stimulated cyclase activity with a monophasic dose-response curve. When assayed together, these stimulations were partially nonadditive, implying the existence of a common adenylate cyclase pool that may be stimulated by both putative neurotransmitters. The dopaminergic antagonist (+)-butaclamol completely blocked dopaminergic stimulation, but had no significant effect on VIP-induced stimulation, indicating that VIP interacts with specific VIP receptor sites, which are distinct from the dopaminergic receptor sites. Furthermore, the specific D-2 dopaminergic receptor agonist LY141865 demonstrated no inhibitory effect on adenylate cyclase activity, suggesting that the interaction between the VIP- and DA-sensitive adenylate cyclase systems does not result from a D-2 receptor-mediated cyclase inhibition in the rabbit retina. Finally, at maximally effective concentrations, DA and VIP were less potent than fluoride or forskolin in the stimulation of cyclic AMP formation, suggesting that adenylate cyclase pools that are not sensitive to DA and VIP may also be present in this retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Guanine Nucleotides on [3H]Glutamate Binding and on Adenylate Cyclase Activity in Rat Brain Membranes

GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg²⁺, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [³H]glutamate binding by 30–40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [³H]glutamate binding occurred in the absence of Mg²⁺. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites.     

Phorbol Esters Increase GTP-Dependent Adenylate Cyclase Activity in Rat Brain Striatal Membranes

Abstract: 4β-Phorbol 12-myristate 13-acetate (PMA), added to a lysed mitochondrial fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of ～30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PMA, respectively. The PMA stimulation is GTP dependent, reaching a maximum of ～60% at 50 μ.M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5′-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalin, and R(-)N⁶-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5′-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by l-α-phosphatidyl-l-serine. Like PMA, 4β-phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4β-phorbol and 4β-phorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.     

Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P

Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 +/- 0.33 ng g-1 and 12.45 +/- 0.76 ng g-1 (mean +/- SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (less than 0.1%) were 12.46 +/- 0.47 ng g-1 and 7.20 +/- 0.37 ng g-1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and substance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide.(ABSTRACT TRUNCATED AT 250 WORDS)