The effects of the acute administration of buprenorphine hydrochloride on the release of anterior pituitary hormones in the rat: evidence for the involvement of multiple opiate receptors

Multiple opiate receptor agonists and antagonists have been found to produce different patterns of anterior pituitary hormone release. The present studies examined the pattern of anterior pituitary hormone release produced by buprenorphine. The effects of the kappa agonist ethylketocyclazocine on thyroid stimulating hormone release were also examined. Following buprenorphine, serum levels of corticosterone and luteinizing hormone were not changed while growth hormone release was stimulated in a dose-dependent manner. Prolactin release was stimulated after the lowest dose of buprenorphine while the highest dose induced a fall in serum prolactin. Similar biphasic effects on thyroid stimulating hormone were seen after either buprenorphine or ethylketocyclazocine. The results provide support for the role of multiple opiate receptors in opiate-induced changes in anterior pituitary hormone release.     

Influence of the estrous cycle and prostaglandins on utero-ovarian vein blood flow in the rat

The blood flow rate in the utero-ovarian vein (UOV) has been measured in adult female rats during the different phases of the estrous cycle. It was observed that the blood flow rate in the UOV is high at proestrus and at estrus and low during diestrus days 1 and 2. The intravenous injection of 10 μg PGF_2α or PGE₂ diminishes the blood flow rate in the UOV. The efficacy of the two PGs in reducing blood flow is different in the various phases of the estrous cycle, being maximal during the day of estrus.     

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p > 0.10). PRL levels in ovariectomized rats in which the NIL had been removed surgically (NIL-X) or only visualized (NIL-C) were 3–7 ng/ml 4, 7, and 28 days after surgery (p > 0.10); however, they were slightly higher in NIL-X $\text{vs.}$ NIL-C rats 14 days after surgery (p < 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p < 0.05); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p < 0.05). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either $\text{via}$ secreting hypophysiotropic hormones or $\text{via}$ conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.     

Influence of the estrous cycle and prostaglandins on utero-ovarian vein blood flow in the rat

The blood flow rate in the utero-ovarian vein (UOV) has been measured in adult female rats during the different phases of the estrous cycle. It was observed that the blood flow rate in the UOV is high at proestrus and at estrus and low during diestrus days 1 and 2. The intravenous injection of 10 μg PGF_2α or PGE₂ diminishes the blood flow rate in the UOV. The efficacy of the two PGs in reducing blood flow is different in the various phases of the estrous cycle, being maximal during the day of estrus.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Changes of immunoreactive somatostatin and beta-endorphin content in rat brain after amygdaloid kindling

A possible contribution of brain beta-endorphin and somatostatin to the epileptogenicity established by amygdaloid kindling was investigated in rats. Fourteen male rats were chronically implanted with electrodes placed bilaterally into the amygdala. The rats received 1 sec of electrical stimulation to the left amygdala each day. Generalized seizures were observed on average 10 days after initiation of kindling and the electrical stimulation was continued up to twenty-one days. Two months after the completion of the kindling procedure, each kindled and control rat was killed by microwave irradiation and the brains were dissected on ice into thirteen subregions. Each region was homogenized and centrifuged twice in 0.1 N acetic acid. The supernatant extracts were decanted and stored at - 20 degrees C until assay. Immunoreactive beta-endorphin and somatostatin were measured by radioimmunoassays. There were no significant differences in brain beta-endorphin contents between the two groups. In kindled rats, immunoreactive somatostatin was increased significantly in amygdala, sensorimotor, piriform, and entorhinal cortex. The results suggest that changes in somatostatin may be associated with epileptic susceptibility induced by the electrical kindling procedure.     

The acute and subacute effects of cadmium on calcium homeostasis and bone trace metals in the rat

The effect of acute and subacute administration of cadmium chloride on calcium homeostasis and the trace metal content of the bone was investigated in the male rat. A single subcutaneous injection of cadmium chloride (1.5 mg Cd⁺⁺/kg) produced a decreased plasma concentration of calcium and a decrease in the femur concentration of both calcium and zinc. Repeated administration of cadmium chloride (1.5 mg Cd⁺⁺/kg daily, for 28 days) caused a marked hypocalciuria that persisted throughout the period of cadmium treatment. There was an accompanying increased excretion of alkaline phosphatase into the urine, and plasma inorganic phosphate was also elevated in these animals. Both of these effects are considered to be evidence of kidney damage.A possible mechanism for this cadmium-induced effect may involve a disturbance of the renal biotransformation of vitamin D, and decreased bioavailability of the essential trace metals due to metallothionein synthesis and excessive loss into the urine.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Target size analysis of serotonin 5-HT1 and 5-HT2 receptors in bovine brain membranes

Freeze-dried crude synaptic membranes prepared from bovine cerebral cortex and striatum were exposed to high energy gamma ray from the source of 60Co. The size of serotonin 5-HT1 receptors labeled by [3H]serotonin and that of 5-HT2 receptors labeled by [3H]spiperone or [3H]ketanserin was determined by target size analyses. The values were 57,000 daltons, 145,000 daltons and 152,000 daltons for the cerebral cortex and 56,000 daltons, 141,000 daltons and 150,000 daltons for the striatum, respectively. The estimated sizes were deduced by reference to enzyme standards with known molecular masses and which were irradiated in parallel. Our results demonstrate that the molecular entities in situ for 5-HT1 receptors are distinct from those for 5-HT2 receptors, thus supporting data on the existence of two distinct populations of serotonin receptors, hitherto evidenced physiopharmacologically.     

Effect of methamphetamine on the development of acute morphine tolerance and dependence in mice

The effect of methamphetamine on morphine analgesia (tail-flick assay) was studied in non-tolerant mice and in mice made acutely tolerant to morphine following a single injection of 100 mg/kg morphine. The analgesic potency of morphine was increased in non-tolerant and tolerant mice to the same extent by 3.2 mg/kg methamphetamine (3.3 and 4.4 fold increases, respectively). In contrast, the ED50's for morphine analgesia and naloxone-precipitated jumping in mice pretreated with either 100 mg/kg morphine or both morphine and 3.2 mg/kg methamphetamine were not significantly different, indicating that methamphetamine had no effect on the development of acute morphine tolerance and dependence. Although methamphetamine had no effect on the development of acute tolerance to morphine, 4-day pretreatment with methamphetamine produced cross-tolerance to morphine analgesia. However, cross-tolerance to morphine was not accompanied by enchanced sensitivity to naloxone.     

Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase

Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.     

Progesterone interaction with estrogen and antiestrogen in the rat uterus--receptor effects.

Daily injections of estradiol or the antiestrogen tamoxifen initially stimulate uterine weight increase and progesterone receptor synthesis, though continued tamoxifen fails to maintain the increased weight. The stimulatory actions of both estradiol and tamoxifen are inhibited or reversed by a single injection of progesterone. It has been hypothesized that progesterone antagonizes estrogen action by reducing estrogen receptor levels, but in the present experiments neither cytoplasmic nor nuclear estrogen receptor was affected. We conclude that progesterone acts at a point beyond estrogen receptor availability or translocation to antagonize estrogen action.     

Distribution of SCEs in lymphocytes in persons with normal,slightly increased,and heavily increased SCEs

The distribution of SCEs in lymphocytes was examined for 165 healthy persons (58 non-smokers and 107 smokers with cigarette consumption ranging from 1 to > 20 per day), and for 1 patient treated with melphalan, a cytostatic drug.The data from the healthy persons did not follow a Poisson distribution. A mixed Poisson that allowed diferent λ values for the 30 cells scored from each person and postulated a gamma distribution for the λs within the 30 cells fitted all the data examined including those from the melphalan-treated patient. In the latter case the 7 samples taken at various times after the treatment could all be represented satisfactorily with a common parameter, c, in the gamma distribution for the λs, even though the mean SCEs/cell varied from 9.8 to 36.8. Because the c parameter determines the spread of λ values within the 30 cells, this suggested that the effect ofthe cytostatic drug was to increase all the σs by a constant amount.The sum of the SCEs taken over all 30 cells in a sample is a convenient summary statistic, and the transformation y = √s + √s + 1 behaves as a normal variate with a constant variance within a group.     

Opioid-specific recognition sites of the mu- and the delta-type in rat striatum after lesions with kainic acid

The binding of 3H-dihydromorphine (3H-DHM) and of 3H-D-ala2, D-leu5-enkephalin (3H-DADL), which are regarded as relatively selective ligands for mu- or delta-type opioid receptors, respectively, was estimated in total particulate fraction of the striatum of rats in vitro, either in tissue of rats after striatal chemolesions with kainic acid or in control rats (not operated or saline injected into the striatum). Kainate lesions reduced the Bmax values of 3H-DHM by about 78 - 88% depending on the method of calculation, and of 3H-DADL by greater than 90%. Furthermore they lowered the Kd-values, suggesting an increase in affinity. The results are discussed with regard to recent hypotheses on the structure and function of opioid receptors.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Formation of hydroxyl radicals from NADH and NADPH in the presence of copper salts

The interaction of copper salts with NADH or NADPH in the presence of hydrogen peroxide at physiological pH is shown to produce hydroxyl radicals. The physiological significance of these results is discussed.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.