Strain-dependent differences in responses to chronic administration of morphine: lack of relationship to brain catecholamine levels in mice.

When measured for weight loss, mortality and degree of physical dependence, 4 strains of mice exhibited widely differing sensitivities to chronically administered morphine. However, no obvious relationship existed between the pharmacological responses to morphine and the steady-state levels of either norepinephrine or dopamine in brain striatal sections of the strains tested. In addition, the injection of naloxone into morphine-dependent mice, which elicits withdrawal jumping, brought about an increase in dopamine levels in the striatal sections of only 1 of the 3 strains tested. Thus, the naloxone-precipitated withdrawal jumping response may not be associated with an elevation of brain dopamine levels.     

Cholinergic agents: antinociception without morphine type dependence in rats

We have confirmed the work of others showing that loss in body weight is a predictable and consistent sign of opiate withdrawal in rats. Rats that were treated chronically with either oxotremorine or physostigmine displayed no weight loss or other signs of opiate-like withdrawal when the drugs were withdrawn. Furthermore, there was no difference in weight loss between morphine dependent rats substituted with saline and those substituted with either cholinergic drug. However, we did observe an increased mortality among rats substituted with a cholinergic agent compared with saline. Rats infused with a mixture of morphine plus oxotremorine or morphine plus physostigmine showed less weight loss, but not fewer behavioral signs, after the end of the infusion than rats treated only with morphine. It is concluded that the cholinergic agents did not cause a morphine-like physical dependence themselves, but appeared to antagonize to some extent the development or manifestation of opiate dependence.     

Differences in the stress response of prolactin in young and aged female rats

The drawing of blood by orbital sinus puncture (OSP) under ether anesthesia is known to produce a marked increase in serum prolactin (PRL levels in young cycling female rats. The effect of this stressful procedure on PRL release was compared in young and aged female rats. Nonstressed PRL levels were obtained from blood drawn by decapitation. Whereas OSP with a one-minute ether exposure induced a marked increase in PRL levels in young rats on all days of the estrus cycle, older cycling female rats on the day of diestrus -1 and aged rats exhibiting prolonged diestrus (PD) showed virtually no increase above nonstressed levels. However, increasing the ether exposure time to five minutes did produce a rise in PRL levels. Old cycling female rats on the day of estrus and aged rats exhibiting constant estrus (CE) did show a PRL increase comparable to that seen in young animals. Ovariectomy (OVX) completely abolished the stress response seen in aged CE rats. The response, though markedly decreased, was still present in young ovariectomized rats. These experiments show that the stress-induced rise in PRL promoted by OSP under either anesthesia is markedly diminished in aged rats exhibiting a diestrus state. The attenuated response seen in these rats is believed due to factors characteristic of the diestrous state of aging.     

Discriminative effects of morphine administered intracerabrally in the rat

Rats were trained in a two-choice discrete trial avoidance paradigm to discriminate between saline and 3.0 mg/kg of morphine administered S.C. The microinjection of 0.3–3.0 μg of morphine into the lateral ventricle produced discriminative effects equivalent to those of the systemic training dose as measured by responding on the morphine-appropriate choice lever. Discriminative effects equivalent to those of the morphine training dose were not consistently produced by administration of morphine into the periaqueductal gray, lateral septum or dorsomedial thalamus in doses as high as 10 μg. However, the discriminative effects of systematically administered morphine were blocked by 10–30 μg of naloxone administered intracerebrally at all of the brain sites tested. Thus, the primary site at which morphine acts to produce discriminative effects in the rat is central, although the specific brain areas mediating these effects remain unidentified. The actions of naloxone could be the result of diffusion of the drug into the ventricular system or into the systemic circulation.     

Endocrine influences on the actions of morphine: IV. Effects of sex and strain

The antinociceptive and temperature responses to morphine were compared in male and female rats from two different strains. Males of both the Sprague-Dawley and Wistar-Furth strains were slightly more responsive to the acute actions of morphine than were females of the same strain. However, Wistar-Furth animals required approximately twice the dose of morphine to display equivalent antinociceptive responses and four times the dose of display equivalent hypothermic responses when compared with Sprague-Dawley animals. During chronic morphine treatment, the development of tolerance was slightly more rapid in males than in females and in Sprague-Dawley animals than in Wistar-Furth animals. Gonadal hormones also influenced morphine responses. Ovariectomized rats were significantly more responsive acutely to morphine and developed tolerance less rapidly than estradiol-treated females. However, alterations of gonadal hormones in males did not affect morphine responses. These results indicate that morphine responses vary considerably between strains of animals and are influenced by gonadal hormones of females, but not of males.     

Effect of corticosterone on the prolactin response to psychological and physical stress in rats

Both corticosterone and prolactin (PRL) levels increase in response to stress. In these studies we examined the effect of corticosterone on the PRL response to both physical (footshock) and psychological (novel environment) stress. Three groups of rats were used: sham adrenalectomized (SHAM), adrenalectomized (ADX), and adrenalectomized with corticosterone replacement (ADX+CORT). The corticosterone-treated animals received 80 ug corticosterone/ml drinking water. Blood samples were drawn via an indwelling cannula and PRL values determined using radioimmunoassay. ADX rats showed a consistently greater PRL response to being placed on a platform above water (novel environment) or when receiving intermittant footshock than did ADX+CORT rats. The PRL response of the latter group was similar to that of the SHAM animals. These findings indicate that corticosterone levels of an animal can significantly attenuate the magnitude of the PRL response to both physical and psychological stress. These findings further emphasize that the PRL response to stress is dependent not only upon the immediate action of the stressor, but also the prior stress history of the animal.     

Variations in myocardial CPK and Na+-K+ ATPase following normo- and hypothermic exposure to dimethyl sulfoxide and glycerol.

Exposure of canine myocardial tissue homogenates to Me₂SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na⁺-K⁺ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me₂SO provides a similar enhancement of Na⁺-K⁺ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me₂SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me₂SO at 0 °C. The results suggest that disruption of the CPK-Na⁺-K⁺ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.     

On the mechanism of erythroid cell sensitivity to chloramphenicol: Studies on mitochondria isolated from erythroid and myeloid tumors

Erythroleukemia mitochondria (E. Mito) and chloroma mitochondria (C. Mito) were isolated from tumors grown in their hosts, DBA/2J mice and Long-Evans rats, respectively. Oxypolarographic tests showed respiratory control and ADP/O ratios typical for well-coupled mitochondria. Therapeutic concentration of chloramphenicol (CAP) had no effect on the energy transfer of those mitochondria. l-[¹⁴C]leucine incorporation into protein was comparable in both types of mitochondria. Although the incorporation at 15 min appeared higher in C. Mito, at 60 min it became similar to that in E. Mito. When CAP was used at the therapeutic concentration of 20 μg/ml about 80% inhibition was observed in both mitochondria. The exogenous amino acid mixture added to the medium was an important determinant in both the rate of leucine incorporation as well as the sensitivity to CAP. Thus, if no amino acids were added the incorporation was reduced to 18–25%. Under these conditions, however E. Mito were significantly more sensitive to the same concentration of CAP than C. Mito. The results suggest that mitochondrial amino acid pool may be involved in the greater sensitivity of erythroid precursors to CAP.     

Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals

Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenopus laevis larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.     

A proposed role for prostaglandins in the modulation of the relaxation response to urotensin I in isolated rat arteries

Urotensin I (UI) elicits dose-dependent relaxation responses in isolated helical strips of rat tail and mesenteric arteries contracted by 10⁻⁵M norepinephrine (NE). The rat mesenteric artery demonstrated a 40 fold lower threshold sensitivity to UI (0.25 mU/M1 versus maximal relaxation at 0.25 mU/m1). Complete relaxation of the rat tail artery with UI could not be achieved, even at doses exceeding 10 mU/m1. Pretreatment of the arterial strips with cyclooxygenase inhibitors had no effect on the contractile response to NE in the tail artery, but reduced NE responsiveness in the mesenteric artery. Significant enhancement of UI relaxation responses in both types of arterial strips was achieved by pre-treatment with the cyclooxygenase inhibiters, suggesting a modulatory role for prostaglandins (PGs) in the expression of the UI relaxation response in NE contracted arterial strips. The major enzymatically formed PG (as assessed by [1-¹⁴C] PGH₂ metabolism in broken cell preparations) in both the rat tail and mesenteric arteries was 6-keto PGF_1α, the stable hydrolysis product of PGI₂. Using a specific RIA to quantify 6-keto PGF_1α release, it was found that UI elicited nearly a two-fold increase in the release of this PG compared to the NE control in both rat tail and mesenteric arteries. These data suggest that PGI₂ may modulate the relaxation response to UI either by direct physiological opposition (PGI₂ elicited contractile response in NE contracted tail and mesenteric arteries at doses exceeding 10−8M) and/or by some as yet undefined mechanism (eg. effects on Ca²⁺, cAMP).     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.     

The mixed lymphocyte response of senescent mice: sensitivity to alloantigen and cell replication time.

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2^d, Mls^b) and DBA (H-2^d, Mls^a) spleen cells served as stimulating cells differing from C57B1/6J (H-2^b, Mls^b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G₂, G₁, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.     

Adrenaline involvement in the presynaptic beta-adrenoceptor-mediated mechanism of dopamine release from slices of the rat hypothalamus

Slices of rat hypothalamus were superfused and endogenous release of dopamine (DA) was measured by high performance liquid chromatography combined with electrochemical detection. The K+ (20 mM)-evoked release in the presence of tetrodotoxin was Ca2+-dependent. The evoked release was facilitated by a beta-agonist, isoproterenol and this effect was completely abolished by a beta-antagonist, 1-propranolol. Isoproterenol also concentration-dependently facilitated the electrically (at 5Hz) evoked release of DA. The pretreatment with 1-propranolol, beta 1-antagonist, atenolol and beta 2-antagonist, butoxamine shifted the concentration-effect curve of isoproterenol to the right. On the other hand, beta 1-agonist, tazolol, beta 2-agonist, salbutamol and low concentration (10(-9) M) of adrenaline also facilitated the release. 1-Propranolol alone reduced the electrically (at 2 Hz) evoked release, and this effect was completely abolished when the adrenaline content in the brain was drastically reduced by use of a potent PNMT inhibitor, DCMB. These findings suggest that in the rat hypothalamus adrenaline released from adrenaline-containing nerve terminals probably modulates DA release via presynaptic beta 1- and beta 2-adrenoceptors on DA nerve terminals.     

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals

The effects of hemicholinium-3 (HC-3) or 4-(1-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 μg HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP resulted in a further reduction in ACh. Pretreatment with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR) against soman was increased from 0.8 to 7.3. These data are consistent with the hypothesis that excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.     

Procoagulant effect of phenobarbital in rats

Daily administration of phenobarbital, 75 mg/kg ip for 4 days, to adult male Sprague-Dawley rats produced a pronounced increase of prothrombin complex activity (PCA) in plasma, i.e. a decrease of the prothrombin time. This effect persisted for 4 to 5 days after the last dose of phenobarbital. The rate constant for the decline of PCA after administration of a PCA synthesis-blocking dose of warfarin was not affected by treatment with phenobarbital but the rate of synthesis of PCA was increased appreciably. Thus, the phenobarbital-induced increase of PCA was caused by increased synthesis of one or more clotting factors.     

Time-dependent loss of prostaglandin E release by adherent cells in response to stimulation by thyroid cells: Prevention of this loss by phytohemagglutinin

Preplating human adherent peripheral blood mononuclear cells (PBMC) for up to 24 hr results in a progressive decrease in their basal PGE release, and in the loss of their ability to increase PGE release during a subsequent 72-hr coculture period with allogeneic human thyroid cells. Phytohemagglutinin (PHA) present during a 24-hr adherent-cell preplating period prevents, in part, the loss of this PGE response to thyroid cells. These data indicate that adherent cells require continual stimulation by the thyroid cells or by PHA in order to maintain their ability to increase PGE secretion in response to thyroid cells.     

Age-related differences in the serum prolactin response during standardized surgery

The aim with the present study was to assess possible age-related differences in the serum prolactin, cortisol and blood glucose responses to standardized surgical stress in humans. Relatively healthy men suffering from inguinal hernias were selected. The subjects were divided into a group of younger people (M=36.4 years, r=13–45, n=7) and one of older people (M=66.5 years, r=56–75, n=9). Surgery was carried out under general anesthesia. Blood was drawn before, during and following the operation. Blood pressure and pulse rate were also monitored. No differences were noticed in plasma prolactin, cortisol, and blood glucose during basal conditions. Even though plasma prolactin increased significantly in both groups during surgery, it was higher in the younger group (M= 56.2 μg/1) as compared with 28.7 μg/1 for the older group, p<.01. Plasma prolactin during surgery, but not under basal conditions, correlated inversely with age. No differences between groups were found during surgery in blood glucose and serum cortisol. This study indicates a diminished stress response in older subjects, possibly due to age-related neuroendocrine changes.     

Vasopressin affects the behavior of rats in a positively-rewarded discrimination task

The effects of subcutaneous injections of vasopressin were investigated in a study utilizing 72 male Long-Evans rats trained in an appetitive black-white discrimination T-maze task. Animals which were reinforced for choosing the black goal arm demonstrated prolonged extinction if they received vasopressin prior to daily extinction sessions. This effect was not observed in animals reinforced for choosing the white goal arm. Prolonged extinction was not found in animals which received vasopresson only during acquisition or in control animals which received saline. Speed and activity scores did not differentiate the groups. These results demonstrate that vasopressin can affect the behavior of rats on a positively-reinforced task.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.