Studies on a possible pituitary effect of monoamines on luteinizing hormone release in ovariectomized rats

Incubation of anterior pituitaries from ovariectomized rats with LHRH and various concentrations of dopamine, norepinephrine or serotonin indicated that none of these neurotransmitters could decrease pituitary LH secretion in response to the releasing hormone. This indicated that the inhibitions of pulsatile LH release previously observed in our laboratory in ovariectomized rats in response to intraventricularly administered catecholamines or stimulation of brain serotoninergic neurons are due to central rather than pituitary effects of these transmitters.     

The roles of the opioid peptides in controlling thyroid stimulating hormone release

We have studied the role of the opioid peptides in controlling TSH secretion. Morphine sulfate significantly decreased, while naloxone had no effect on, basal plasma TSH levels of female rats. In contrast, naloxone blocked the stress-induced fall in plasma TSH. Microinjection of β-endorphin into the third ventricle resulted in a fall in TSH while such injection of naloxone into the posterior hypothalamus increased TSH. Microinjection of β-endorphin directly into the pituitary caused a rise in plasma TSH. It is concluded that opioid peptides probably play no role in basal TSH secretion, but are involved in the stress-induced fall in TSH. Furthermore, it appears that opioid peptides have a site of action in the hypothalamus to decrease TSH and a direct pituitary action to increase TSH.     

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.     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Thyrotropin-releasing hormone and a homologous peptide in the reproductive system of the female rat and pig

TRH and a TRH homologous peptide have been shown to occur throughout the female rat and pig reproductive systems by TRH radioimmunoassay, SP-Sephadex C-25 cation exchange chromatography, and parallel line analysis of the assays. The total amount of TRH and TRH homologous peptide immunoreactivity was highest in the oviducts followed by the ovary and then uterus. The concentration of TRH immunoreactivity in all reproductive organs of the rat fell gradually from one month of age. TRH and the TRH homologous peptide were not parallel on serial dilution and measurement in the same TRH radioimmunoassay. The rapid degradation of TRH by pig follicular fluid may explain the higher measured concentration of TRH homologous peptide compared to TRH not only in pig follicular fluid but also in the pig ovary as a whole.     

Allosteric transitions and membrane-bound ATPase from rat tissues: The effect of fat deprivation on the allosteric inhibition by fluoride

In rats red a fat-sufficient diet, ATPases (ATP phosphohydrolase, EC 3.6.1.3) from heart, kidney and brain microsomes showed allosteric kinetics for the inhibition by F^?, with values ofn = ?2.0. In rats fed a far-free diet, the values ofn for the ATPases changed from ?2.0 to ?1.0 in heart and kidney microsomes. When these animals were then fed a fat-sufficient diet the values ofn reached the control values. In brain microsomal ATPases no modification of the values ofn were found between both groups of animals. The regulatory properties of the membrane on bound ATPases are discussed.     

Dissociation between increased growth hormone and prolactin secretion during the morning hours of early pregnancy in the rat

We investigated whether serum growth hormone (GH) concentration changes in association with the rise in serum prolactin (PRL) concentration known to occur during the early morning hours in the pregnant rat. Animals were kept in a room with the lights on from 0500 to 1900 hours (hr) daily and decapitated for the collection of trunk blood at 2200 or 2400 hr on Day 6 of pregnancy or at 0200, 0400, 0800 or 1000 hr on Day 6 of pregnancy. Serum GH concentration rose more than 4-fold from low levels at 2200 and 2400 hr to higher levels at 0400 and 0800 hr and then declined by 1000 hr. Serum prolactin (PRL) concentration followed a similar pattern except that it returned to low levels earlier, by 0800 hr. Serum luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone concentrations showed no significant changes. Serum GH levels at 0800 hr in pregnant rats were higher than those observed in cyclic rats (13 time periods sampled). The results demonstrate that serum GH concentration is elevated during a circumscribed period in the 6- to 7-day pregnant rat. The time of onset of the rise is similar to that for serum PRL but the elevation in GH levels persists longer than that for PRL.     

Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the third ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of substance P (SP) or neurotensin (NT) and plasma GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of physiologic saline iv or into the third ventricle did not modify plasma hormone levels. Intraventricular injection of SP or NT at doses of either 0.5 or 2 μg elevated plasma GH concentrations within 5 min and they remained elevated for 60 min. Third ventricular injection of similar doses of SP or NT had no effect on plasma TSH. An intermediate dose of 1 μg of SP or NT given iv had no effect on plasma GH but NT elevated plasma TSH. Incubation of hemipituitaries from OVX rats with varying doses of SP or NT did not alter GH release into the medium but TSH release was enhanced with NT at doses of 100 or more ng/ml of medium. It is suggested that SP acts centrally to stimulate growth hormone-releasing factor (GRF) or to inhibit somatostatin release and thereby enhance GH release and that NT acts directly on the pituitary to stimulate TSH release.     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

Assessment of blood platelets as a model for CNS response: comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons

We have previously reported that caffeine significantly enhanced 5-HT uptake and reduced 5-HT release from crude synaptosomal fractions obtained from rat cerebral cortex and from midbrain raphe region. Blood platelets, as reported by many laboratories and also demonstrated in our own labs, have a very active mechanism for 5-HT uptake and storage. In this regard platelets bear a high degree of similarity to brain serotonin neurons. The present experiments were, therefore, carried out to investigate the effects of caffeine on 5-HT uptake and release from rat platelets in an attempt to assess the possibility of using platelets as a model for studying the CNS effects of caffeine. Platelet rich plasma was prepared from the trunk blood of decapitated rats. Effects of caffeine were investigated at 10(-7), 10(-6), 10(-5) and 10(-4)M, on both the high affinity 3H-5-HT uptake and the spontaneous 5-HT release from 3H-5-HT preloaded platelets. The results show that caffeine did not change 5-HT uptake into platelets. In brain synaptosomes the same concentration of caffeine, however, increased 5-HT uptake dose-dependently. The results also revealed that caffeine increased 5-HT release from rat platelets in a concentration-dependent manner. The concentrations 10(-6), 10(-5), and 10(-4)M increased release significantly compared to control. This finding is also in contrast to that observed in synaptosomes of brain serotonin neurons where caffeine decreased 5-HT release. It is concluded, therefore, that the rat blood platelet is not a suitable model for studying these CNS actions of caffeine. Furthermore, our observations imply that rat platelet serotonin uptake and release mechanisms are not identical to those mechanisms in brain serotonin neurons.     

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of ³²P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.     

The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti.

The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti. The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

The presence of the milk protein, alpha-lactalbumin and its mRNA in the rat epididymis

alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.     

The effect of organic compounds on the crystal habit and crystallization of normal and sickle cell hemoglobin in phosphate buffer

Comparative studies were made on the effect of numerous organic compounds in promoting the crystallization of human hemoglobin in 1.9 m phosphate, pH 7.0. It was found that alicyclic or benzenoid structures are essential for promoting crystallization of hemoglobin under these conditions. Hemoglobin crystals prepared in the presence of toluene differed in habit from crystals prepared in its absence. It is suggested that steric factors determine the effectiveness of organic substances in promoting the crystallization of hemoglobin and that the heme group is the binding site involved in the complex formation.The solubility of homozygous sickle cell hemoglobin HbS was found to be less than the heterozygous hemoglobins AS and AC or normal hemoglobin HbA in the presence of organic substances promoting the crystallization of hemoglobin.     

Association of leucogenenol,a thymothyroid hormone,with carrier proteins in the thymus

Leucogenenol a heterocyclic enolic thymothyroid hormone (MW 383) whose concentration in the serum regulates the rate at which already committed cells of the bone marrow develop into functional cells, was found to be associated in the thymus with a carrier protein. The carrier protein for leucogenenol is not precipitated by heating to 80° but following this treatment leucogenenol is precipitated in association with proteins precipitated by acetone and then by saturated ammonium sulfate. On chromatography on Sephacryl G-200 it was found that leucogenenol was associated with proteins of MW approximately 38,000. Leucogenenol is not eluted from the chromatographic column if it is not associated with its carrier proteins. It is suggested that other hormones such as those associated with the reproductive cycle or compounds that result from tissue damage induce the liberation of leucogenenol from its carrier protein in the thymus to the circulation where it is associated as previously described, with a protein of approximately MW 300,000.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

The effect of carbon dioxide on local cerebral blood flow during surface hypothermia in dogs.

Local cerebral blood flows were measured using the hydrogen clearance technique. This was found to be a satisfactory method.During hypothermia, maintenance of an F_ECO₂ above 5% is accompanied by higher LCBF while the opposite occurs with hyperventilation to 3% F_ECO₂.