Effects of desglycinamide9, (Arg8) vasopressin and vasopressin antiserum on the acquisition of intravenous cocaine self-administration in the rat

The effect of the vasopressin neuropeptide desglycinamide9, (Arg8) vasopressin (DGAVP) on the acquisition of intravenous cocaine self-administration was studied. Rats were tested under conditions of reduced body weight in a continuous reinforcement operant procedure, during five daily 3 h sessions. Under these conditions, the rate of self-administration obtained with 0.125 and 0.25, but not 0.063 mg.ml-1 cocaine, exceeded the rate obtained with saline. Daily pretreatment with DGAVP (5 micrograms/rat, s.c.) decreased self-administration of 0.125 and 0.25 mg.ml-1 cocaine to the level obtained with saline, but had no effect on self-administration of 0.063 mg.ml-1 cocaine and saline. Using a similar procedure, it was shown that daily intracerebroventricular pretreatment with vasopressin antiserum significantly increased self-administration of 0.125 mg.ml-1 cocaine, without affecting self-administration of 0.063 and 0.25 mg.ml-1 cocaine and saline. The results support previous findings obtained with vasopressin neuropeptides in drug self-administration studies and suggest that DGAVP decreases the acquisition of cocaine self-administration by attenuating the reinforcing effects of cocaine and that endogenous vasopressin may be involved in the acquisition of cocaine self-administration.     

Intraventricular administration of antivasopressin serum inhibits retention in mice

Elevations and decrements in the levels of the posterior pituitary hormone vasopressin result in facilitations and deficits in retention, respectively, in rats. Despite the frequent use of mice in studies of pharmacological influences on retention, there is a paucity of information regarding the role of endogenous peptides, particularly vasopressin, in the memory processes of mice. In the present experiment, mice suffering from acute inactivation of central vasopressin, induced by an immediately posttraining, 2 microliters, intracerebroventricular injection of antivasopressin serum, displayed a retention deficit for passive avoidance training. The results of this experiment suggest that endogenous vasopressin modulates the memory processes of mice, as well as rats.     

Neurohypophyseal principles and memory.

The neurohypophyseal hormones vasopressin and oxytocin modulate memory processes. Vasopressin facilitates, while oxytocin attenuates memory consolidation and retrieval. These influences are located in different regions of the molecules. Thus, the neurohypophyseal hormones act as precursor molecules for neuropeptides involved in memory processes. The covalent ring structures of both vasopressin and oxytocin mainly affect consolidation; the linear parts, retrieval processes; while nearly the whole oxytocin or vasotocin molecule is needed for attenuation of consolidation and retrieval. Regional studies, utilizing microdissection techniques in combination with a sensitive radioenzymatic catecholamine assay, revealed a distinct pattern of effects on cerebral alpha-methyl-p-tyrosine methylester-induced catecholamine disappearance following intraventricular vasopressin administration in limbic midbrain structures. In situations in which the amount of bioavailable vasopressin in the brain is absent, as is the case in the Brattleboro rat with hereditary diabetes insipidus, or neutralized in normal Wistar rats following the intraventricular administration of antivasopressin serum, regional catecholamine disappearance in most cases is altered in a direction opposite to that observed after intracerebroventricular vasopressin administration. These results indicate that vasopressin modulates memory processes by modulation of neurotransmission in distinct catecholamine systems. Recent experiments suggest that the influence of vasopressin on memory consolidation is mediated by the dorsal noradrenergic bundle via terminal regions of this bundle.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

Vasopressin receptor distribution in adrenalectomized rats using vasopressin anti-idiotype

Following adrenalectomy, it has been demonstrated that parvocellular corticotropin-releasing factor-containing neurons in the paraventricular nucleus (PVN) of rat hypothalamus synthesize vasopressin. The present study examined whether putative vasopressin receptors are expressed in parallel with the appearance of vasopressin immunoreactivity in these parvocellular neurons. A vasopressin anti-idiotypic antibody which immunostains putative vasopressin receptors associated with magnocellular PVN neurons was utilized. Following adrenalectomy, antivasopressin immunostained neurons in parvocellular and magnocellular PVN, whereas the anti-idiotypic antibody immunostained magnocellular neurons only. We therefore conclude that the putative vasopressin receptor recognized by the anti-idiotype is not demonstrated in association with parvocellular vasopressin-producing neurons of the adrenalectomized rat.     

A non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

A simplified method for evaluating serum dehydro-epiandrosterone (DHEA) without chromatography has been developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ⁵ antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μ1) was extracted with 1 ml of n-hexane. One milliliter of 80% methanol was added to the n-hexane extract which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- ³H. The accuracy, precision, sensitivity and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use.     

beta-Endorphin controls vasopressin release during foot shock-induced stress in the rat

This study tested the possibility that beta-endorphin is involved in the regulation of vasopressin release during stress induced by inescapable electric foot shock. To this end, a specific anti-beta-endorphin antiserum or a control serum lacking the specific anti-beta-endorphin antibodies was administered to male rats. Plasma vasopressin concentrations, measured by radioimmunoassay, were not affected by brief foot shock stress in control rats, but were raised significantly by the stress in animals which had received an intracerebroventricular (i.c.v.) injection of the anti-beta-endorphin antiserum. In contrast, when the same volume of the anti-beta-endorphin antiserum was injected into a tail vein, foot shock stress produced only a slight effect on vasopressin release. I.c.v. injection of the antiserum changed neither basal nociceptive threshold nor stress-induced analgesia as revealed by the tail-flick latency. Vasopressin release induced by an osmotic stimulus was not influenced by the anti-beta-endorphin antiserum given i.c.v. The opiate antagonist naloxone or the glucocorticoid dexamethasone raised plasma vasopressin concentration in stressed rats which had received the control serum (i.c.v.); however, after i.c.v. injection of the anti-beta-endorphin antiserum neither naloxone nor dexamethasone elevated the plasma vasopressin concentration beyond the level reached by the anti-beta-endorphin antiserum (i.c.v.) alone. These results suggest that beta-endorphin inhibits the release of vasopressin during foot shock-induced stress in the rat.     

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These ''self-administration'' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior^1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction^3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher^10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration ^12,16.Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)^17-22. Auditory and visual stimuli are self-administered by several species^18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement²⁴. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS²⁴. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation ²⁵ is not observed in this model²⁴. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose²⁶. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked.In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.Download video file.^{(54M, mov)}     

A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model

Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-l-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N ¹-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N ¹-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.     

An antiserum against a glycoprotein functional in flagellar adhesion between Chlamydomonas eugametos gametes

Chlamydomonas eugametos gametes agglutinate sexually by their flagellar surfaces. The agglutination factor on mating type minus (mt^-) gametes is thought to be a glycoprotein named PAS-1.2. To test this idea, an antiserum was raised against purified PAS-1.2., which reacted with isolated PAS-1.2 (immunoprecipitation tests) and blocked the ability of isolated PAS-1.2 to induce sexual twitching in mt ⁺ gametes. When tested with living cells, the antiserum specifically agglutinated mt ^- gametes and induced a reaction resembling twitching. Mt ⁺ flagella were shown to bind the antiserum (indirect immunofluorescence) but much less than mt ^- gametes. Mt ^- gametes pretreated with Fab fragments of the antiserum were unable to reproduce sexually, while treated mt ⁺ gametes were unaffected. This effect presumably results from the ability of the serum to block mt ^- sexual agglutination, for mt ^- isoagglutinin was completely inactivated by the serum, while mt ⁺ isoagglutinin was unaffected. It is therefore argued that PAS-1.2 is the in vivo mt ^- agglutination factor. However it is shown that the antiserum was able to react in vitro not only with PAS-1.2 but with several other proteins in both mt ^- and mt ⁺ flagella.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid-Schiff - GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PBS phosphate buffer-saline - Fab univalent antibody fragment The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Luteinizing hormone-releasing factor (LRF)-like immunoreactivity in rat pancreatic islet cells.

Luteinizing hormone-releasing factor (LRF)-like immunoreactive material was demonstrated by the three-layer immunoperoxidase method in formalin-fixed tissue sections of the rat pancreas. Anti-LRF antiserum was prepared in rabbits by immunizing with synthetic LRF coupled to bovine serum albumin (BSA). The immunoreactive site of LRF reacting with antiserum resided between residues Tyr⁵ and Gly¹⁰-NH₂. A positive staining reaction was observed in the islet cells with the use of anti-LRF antiserum after solid phase immunoadsorption with BSA, whereas no staining was observed when adjacent control sections were prepared with anti-LRF antiserum after immunoadsorption with an LRF-BSA conjugate, or with rabbit anti-oxytocin antiserum. LRF-like immunoreactive material was isolated from the rat pancreata by methanol extraction. This material coeluted with synthetic and hypothalamic LRF in cation exchange chromatography on carboxymethyl cellulose, and dilutions of it gave an inhibition curve parallel to that of synthetic LRF in radioimmunoassay. The concentration of LRF-like material in the rat pancreas is 1.1 pg/mg wet weight. These results suggest that LRF or a closely LRF-related peptide is shared by the central nervous system and the gastrointestinal tract.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat

Summary An immunoelectronmicroscopic method for the specific localization of neurohypophyseal hormones was developed in neurohypophyses of Wistar and Brattleboro rats, the latter strain being homozygous for diabetes insipidus. If the proper precautions were omitted, a marked cross reactivity between antivasopressin and antioxytocin preparations was found. Cross reaction of an antivasopressin plasma with oxytocin, at a dilution of less than 11600, resulted in electron density of all granules within neurosecretory fibres of the Brattleboro and Wistar neurohypophyses. However, this cross reactivity could be eliminated either by sufficient dilution of the antiplasma, or by its purification. Purification of the antibodies was performed by absorption to agarose beads coated with the cross reacting component. Upon incubation with antivasopressin (diluted unpurified 11600 or purified 180) and unpurified antioxytocin (1400) plasma, sections of a Wistar neurohypophysis revealed two types of neurosecretory fibres, containing either electron dense or lucent granules. Oxytocin and vasopressin containing neurosecretory fibres were found as clusters in the neurohypophysis. The specificity of both unpurified antivasopressin (11600) and antioxytocin (1400) plasma was confirmed on serial sections of a Wistar neurohypophysis, alternately incubated with the solutions of the two antibodies.These data prove that the one-cell-one-hormone hypothesis holds true for the hypothalamic-neurohypophyseal system.The authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group on neuroendocrinology for their suggestions and critical remarks, and Mrs. M. Mud, Mr. P. Wolters and Mrs. A. van der Velden for their skilful assistance     

A radioimmunoassay for serum 11-deoxy-17-ketosteroids

A simplified method for evaluating serum 11-deoxy-17-ketosteroids (11-deoxy-17-KS) equivalent to dehydroepiandrosterone sulfate (DHEAS) has been developed without solvolysis and chromatography. 5μl of serum or plasma was added to 1 ml of ethanol, mixed, and centrifuged. 10 or 20 μ1 of the supernatant was evaporated to dryness and incubated with anti-11-deoxy-17-KS antiserum obtained by immunizing a rabbit with DHEA-3·O·CO-BSA which was prepared from DHEA-3·O·COC1 and containing DHEAS-7α³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEAS-7α³H. The accuracy, precision and sensitivity were satisfactory. The blank values could not be differentiated from zero. As the antiserum reacted not only on DHEAS but also on androsterone sulfate and etiocholanolone sulfate, serum 11-deoxy-17KS obtained by the radioimmunoassay expressed nearly the sum of 100% of DHEAS, 45% of androsterone sulfate and 35% of etiocholanolone sulfate in the serum. A good correlation was found between serum 11-deoxy-17-KS and DHEAS obtained by the radioimmunoassay described in a preveous paper (1). The present radioimmunoassay is the simplest method for the evaluation of the concentrations of C₁₉ steroids in the serum.     

Selective Inhibition of Macromolecular Synthesis in <Emphasis Type="Italic">Pasteurella septica</Emphasis> by Antiserum and its Reversal by Iron

EVIDENCE from work with both in vivo and in vitro systems suggests that the ability of an organism to acquire iron from serum transferrin may be an essential feature of pathogenicity^1–4. Conversely, the evidence suggests that the ability of the host to interfere with this reaction may constitute an important means of defence^{1, 2}. Specific antiserum and a heat labile factor in fresh serum, possibly complement, have been implicated in the bacteriostatic and bactericidal action of sera on Pasteurella septica¹. Both these effects can be abolished by saturating the serum transferrin with iron. A start has now been made in investigating the biochemistry of these processes.     

Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [³H]glycerol, [³H]myristate, [³H]choline or [³H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [³H]glycerol or [³H]myristate were stimulated by vasopressin, no significant decrease in the labelling of phosphatidylcholine was detected, but the labelling of phosphatidic acid was elevated. However, the labellings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [³H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells.Abbreviations BHK-21 cells baby hamster kidney-21 cells     

Blocking of Lymphocyte-mediated Cytotoxicity for Rat Hepatoma Cells by Tumour-specific Antigen-Antibody Complexes

LYMPHOCYTES from tumour-bearing animals are often cytotoxic in vitro against cultured tumour cells from the same individual^1–4. It is possible that the serum of tumour-bearing hosts may contain circulating factors which interfere with the cell-mediated immune responses concerned in tumour rejection reactions⁵. Evidence has been provided by the demonstration that lymphocyte cytotoxicity against cultured tumour cells could be blocked by first exposing tumour cells to serum from tumour-bearing animals^2,3; similar effects have also been observed in cancer patients^6,7. The blocking factor in tumour-bearer serum has the characteristic properties of 7S immunoglobulins², suggesting the involvement of tumour-specific antibody. Serum blocking activity is rapidly lost, however, in animals rendered tumour free and the activity of tumour-bearer serum can be neutralized by the addition of serum from these animals^8,9. One explanation is that the blocking factor in tumour-bearer serum is antigen-antibody complex and the objective of these studies, using a transplanted rat hepatoma (D23), was to test directly whether such complexes prepared from solubilized tumour-specific antigen and antiserum exhibit blocking activity.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-125I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

CHARACTERIZATION OF THE BASIS FOR DIFFERENCES IN SERUM DBH ACTIVITY IN IMMATURE AND ADULT RATS BY USE OF HOMOLOGOUS ANTIBODY

Abstract– Rat serum dopamine-β-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD₅₀) decreased 5-fold from 0.25 to 0.05 μl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent K_m for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme. To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immunotitration of DBH activity in vivo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2μl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 ± 0.6 days; the estimated fractional rate of degradation was 0.23 ± 0.06 day^?1 and the rate of entrance was 2.3 ± 0.2 units/ml/day. After treatment of 4-day-old rats with 1 μl of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 ± 0.5 days: the estimated average fractional rate of degradation was 0.22 ± 0.06 day^?1 and the average rate of entrance was 10.7 ± 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats.     

Antibodies to pig kidney (Na++K+)-ATPase inhibit the Na+ pump in human red cells provided they have access to the inner surface of the cell membrane

Antisera from rabbits that had been immunized with a highly active membrane preparation of (Na⁺ + K⁺)-ATPase from the outer medulla of pig kidney strongly inhibited (Na⁺ + K⁺)-ATPase activity in various tissues. When the antiserum was incorporated into released human red cell ghosts, the ouabain-sensitive efflux of Na⁺ into both 15 mM K⁺ and K⁺-free high Na⁺ media was completely abolished. This effect was not observed when non-immune serum was used, or when the immune serum was allowed access only to the outer surface of the red cell membranes.