Substance P release from spinal cord slices by capsaicin

The release of substance P (SP) from slices of hypothalamus, substantia nigra and spinal cord of the rat was studied. In contrast to 47 mM potassium, capsaicin induced SP release from spinal cord slices only. The SP release was not reduced by 10^?6 M tetrodotoxin but was abolished by a Ca²⁺ free medium containing 3 mM ethylene-glycol-bis- (β-aminoethyl-ether) N,N-tetraacetic acid (EGTA). Capsaicin induced SP release was dose-dependent and exhibited tachyphylaxis. The ability of capsaicin to release SP supports the hypothesis that SP is involved in pain transmission and in neurogenic plasma extravasation.     

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain.

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.     

Diurnal variations in learning performance in chickens

Diurnal variations in the learning performance of young chicks were investigated using a visual discrimination task which requires birds to discrminate grains from a background of pebbles. Chicks accustomed to receiving fresh food daily in the morning were found to learn well during the day, in that they pecked almost exclusively at grains; but during the night they pecked indiscriminately at grains and pebbles. This occurred even though food was available ad libitum. Chicks accustomed to receiving fresh food daily in the evening learnt the task during the day, and also late at night. Thus the shape of the performance cycle depends in part on environmental factors. Other factors, such as activity, which may contribute to, or co-vary with, this variation in learning performance were investigated.     

Opioids modulate periodicity rather than efficacy of peristaltic waves in the guinea pig ileum in vitro

The influence of opioid receptor blockade by naloxone and opioid receptor activation by opioids on peristalsis was studied in isolated segments of the guinea pig ileum.1. (-)Naloxone, but not (+)naloxone, increased the mean number of peristaltic waves per min within periods of elevated intraluminal pressure. Naloxone tended to modify intermittent peristalsis into ongoing peristalsis, whereas opioids worked in an opposite fashion. 2. Maximum amplitudes of luminal volume displacement during single peristaltic waves were not decreased by opioids. (-)Naloxone, however, applied to non-pretreated segments, decreased transitorily the efficacy of single peristaltic waves to a small, but statistically significant degree 3. Enhancement of peristalsis by naloxone decreased over time, although enough naloxone was present to occupy all opioid receptors. This suggests that opioid receptor blockade induces some compensatory mechanism.     

Verapamil is a competitive inhibitor of gamma-aminobutyric acid and calcium uptakes by mouse brain subcellular particles

We found that verapamil and its methoxy analogue, D600, were relatively potent (micromolar) inhibitors of Na+-dependent GABA uptake by a mouse brain microsomal subfraction (P3). Verapamil was competitive with GABA and uncompetitive with Na+ in the uptake assay with the P3 fraction. These substances were much less effective in inhibiting GABA binding in a receptor-related assay system with synaptosomal membranes. Inhibition by verapamil of Na+-dependent 45Ca++ uptake by the P3 particles was competitive with Ca++. A consideration of our results with those in the literature led to the suggestion that the interaction of verapamil and related substances with GABA and 45Ca uptake processes by the P3 fraction, as well as with many other membrane activities, may be allosteric in nature rather than directly competitive with specific ligands at their binding sites.     

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10^?5M) had no effect on contractile tension or on the PIE to isoproterenol (10^?10?10^?7M) or ouabain (10^?6?10^?4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10^?4?10^?6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10^?8M), by dantrolene (10^?6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.     

Fixed interval and fixed time treadle pressing in the pigeon: A comparison with FI and FT keypecking

Experiment I used non-naive pigeons having previously performed on both keypecking and treadlepressing Fixed Interval schedules. In condition IT, treadlepressing was reinforced on successive Fixed Interval 60 seconds, Fixed Time 60 seconds and Fixed Interval 60 seconds schedules. Subsequently (condition IK), the same subjects pecked a key on an identical schedule sequence (FI60, FT60, FI60). In Experiment II, separate groups of naïve subjects were assigned either to treadlepressing (condition IIT) or keypecking (condition IIK) and to the same schedule sequence (FI60, FT60, FI60). Treadle pressing and keypecking decreased greatly in Fixed Time schedules. Curvature indices, pauses and running rates were less sensitive than response rates to the switching from one schedule to the other. Experiments I and II yielded similar results, experimental history accounting only for minor differences. The results were discussed in relation to interspecies differences in the temporal regulation of behavior and operant versus respondent control of the response and schedule-induced behaviour.     

The depolarising action of capsaicin on rat isolated sciatic nerve

The effect of capsaicin on the isolated sciatic nerve of rat was studied by extracellular recording of membrane polarisation. Capsaicin depolarised the sciatic nerve, but desensitization occurred rapidly upon repeated administration. Several other neuroactive substances, including substance P, were inactive. The depolarisation was reduced in nerves depleted of unmyelinated fibres by neonatal capsaicin treatment, suggesting that it occurs mainly in C-fibres. This depolarising action of capsaicin could explain the irritant and acute antinociceptive properties of capsaicin.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracelluar pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-³H-thymidine incorporation into egg DNA. Removing ammonia decreases NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.     

Contact sensitivity in rabbits sensitized with dinitrophenylated Mycobacterium tuberculosis

The conjugation of Mycobacterium tuberculosis with DNFB results in the formation of a haptenated preparation that induces the formation of contact sensitivity when administered subcutaneously. This contact sensitivity can be measured in vivo by topical application of the free chemical and in vitro by lymphocyte transformation. The antigens suitable for the in vitro detection are those preparations obtained by the haptenation of cell membranes. Haptenation of serum proteins, of homologous and heterologous origin, does not produce antigens suitable for in vitro assay. The antigen requirements for the in vitro transformation assay of contact sensitivity are similar for adjuvant induced sensitivity as well as for free chemical induced sensitivity.     

Implantation-associated proteinase in mouse uterine fluid

Proteinase activity was detected in mouse uterine fluid by means of a new casein-substrate assay. The activity was found to be generally low in diestrous and proestrous stages of the estrous cycle and was more variable in proestrous. During pregnancy, activity was very low on day 1 (counting the plug date as day 0). By day 3, proteinase activity (expressed as Pronase equivalents/mg protein of uterine fluid) increased more than 100-fold, and then declined on day 4. Peak activity thus coincides with initiation of embryo implantation, which occurs on day 3 of pregnancy in the strain tested. The results provide direct biochemical support for previous indirect bioassay indications of the presence in uterine fluid of a proteolytic factor of uterine origin. The quantitative changes observed here are also consistent with previous bioassay observations and with the hypothesis that the uterine proteinase may mediate initial attachment of the blastocyst to the uterine wall. These and other data are used to formulate a 2-stage hypothesis of implantation, according to which uterine and trophoblast proteinases act sequentially to cause attachment and invasion, respectively.     

Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range

Conditions and simple precautions are presented for carrying out highly reproducible and sensitive peptide mapping by thin-layer chromatography and subsequent electrophoresis of subnanomole amounts of tryptic digest on silica gel G or GHL plates. The fluorogenic reagent “fluorescamine” is employed for visualization under long-wavelength ultraviolet illumination. Permanent photorecording of high-contrast images, using readily available filters, is substituted for subjective hand scoring of plates. Contrast reversal is used to produce peptide maps suitable for half-tone reproduction.     

Difference in androgen-dependent change of non-histone proteins between dorsolateral and ventral prostates of rats

A novel species of non-histone protein having a molecular weight of approximately 20,000 (20K), abundantly localized in the dorsolateral prostate, was found to be decreased in the content by castration and to be restored by replacement of androgen, in addition to non-histone proteins of molecular weights ≥ 34,000. The content of $\text{20K}\text{DNA}$ was more rapidly decreased by castration, but more slowly restored by replacement of androgen, with the dorsolateral prostate than the ventral prostate. Of other nuclear proteins, non-histone proteins of molecular weights ≥ 90,000 in the dorsolateral prostate were more susceptible to the decrease by castration, whereas those of all kinds of histones were hardly dependent on the androgen level.     

Defective phospholipid metabolism in the retinular cell membrane of norpA (no receptor potential) visual transduction mutants of Drosophila

The phosphorylation of photoreceptor phospholipids in the three alleles of Drosophila visual mutants (norpA: no receptor potential A gene) was studied. In the normal strain, the gamma-32P of ATP was transferred mainly to phosphatidic acid (PA) and diphosphoinositide (DPI), while, in the mutants, we found that the phosphorylation of PA was drastically reduced, but that of DPI was not. The radioactivity incorporation into PA closely parallels with the degree of the mutant genes' expressivity among the three alleles of norpA tested. Therefore, the abnormality found in the phosphorylation of diglycerol to PA may be closely related to the primary mutant defect in the phototransduction mechanism.     

Anticonvulsive effects of endogenous prostaglandins formed in brain of spontaneously convulsing gerbils

Seizures were induced in a strain of epileptic gerbils by moderate environmental stress. Concentrations of five different cyclooxygenase products (PGD₂, PGF_2α, PGE₂, 6-keto-PGF_1α and thromboxane B₂) were measured in brain by specific radioimmunoassays before and at different time intervals after the onset of clonic-tonic convulsions. All prostanoids markedly increased subsequent to the convulsions. Maximal concentrations were reached after about 15 min. The major compound detected was PGD₂, followed by PGF_2α and lower concentrations of the other cyclooxygenase products. Indomethacin completely prevented the convulsion-induced formation of prostanoids. Fifteen min after a first seizure almost all animals proved to be protected against a second convulsion. Indomethacin pretreatment markedly reduced the number of convulsion-resistant animals. These findings are compatible with the hypothesis that endogenous prostaglandins exert anticonvulsive effects.     

Role of substance P in water homeostasis.

The effect of the naturally occurring peptide substance P on release of antidiuretic hormone (ADH) was studied in anesthetized dogs. Intravenous infusions of substance P in doses of 0.5, 5.0, and 50 ng/kg/min increased plasma concentrations of ADH in a dose-related fashion. At the two low doses, this increase occured in the absence of changes in urine volume, sodium excretion, free water clearance, and urinary cyclic AMP excretion. In addition, when substance P was administered in a concentration of 0.5 ng/kg/min, plasma levels of ADH were increased even though blood pressure did not change, suggesting that substance P may release antidiuretic hormone by a direct mechanism. Intrarenal infusions at a rate of 0.5 and 5 ng/kg/min caused dose-related decreases in free water clearance and significant increases in urinary cyclic AMP excretion. These data suggest that substabce P may play an important role in the regulation of water balance.     

Stimulation of prostaglandin E2 synthesis in chondrocytes by a factor derived from activated macrophages

The serum-free spent medium of lipopolysaccharide-activated rabbit peritoneal macrophages contains a proteinaceous factor that stimulates the synthesis of PGE₂ in rabbit articular chondrocytes. Synthesis of this factor by macrophages is inhibited by cycloheximide. Stimulation of PGE₂ in chondrocytes is detected after a four-hour exposure to the macrophage factor and is completely abolished by the addition of either cycloheximide or indomethacin to the chondrocyte cultures. The macrophage derived factor has an apparent molecular weight of 30,000, is heat stable and not inactivated upon reductive alkylation or on treatment with phenylglyoxal. Activity is partially destroyed upon treatment with acid (pH 2.0) and upon trypsin treatment.     

Arginine8-vasopressin affects catecholamine metabolism in specific brain nuclei

Following the i.c.v. administration of arginine⁸-vasopressin (30 ng in 1 μl saline) to rats that had been injected i.p with α-MPT 30 min prior to the administration of the peptide catecholamine metabolism was altered in a restricted number of brain nuclei. Noradrenaline disappearance was accelerated as compared to saline treated controls in the dorsal septal nucleus, the anterior hypothalamic nucleus, the medial forebrain bundle, the parafascicular nucleus, the dorsal raphe nucleus, the locus coeruleus, the nucleus tractus solitarii and the Al-region. In the supraoptic nucleus and the nucleus ruber a decreased noradrenaline disappearnce was observed after the administration of the peptide. Dopamine disappearance was accelerated in the caudate nucleus, the median eminence, the dorsal raphe nucleus and the A8-region. These results support the view that vasopressin is participating in the regulation of a variety of physiological processes by modulating neurotransmission in specific brain nuclei.     

Synergistic interaction between prostaglandins and paf-acether in experimental animals and man

Platelet activating factor (PAF-acether) is released from a variety of inflammatory cell types and has properties appropriate to a mediator of allergy and inflammation. Here, we have examined the interaction between PAF-acether and the prostaglandins, PGE2 and ZK 36374 (a stable analogue of prostacyclin, PGI2) in the skin of guinea-pigs and human volunteers. PGE2 and ZK36374 significantly potentiated increased plasma protein extravasation induced by PAF-acether in guinea-pigs, assessed by extravasation of I-125-HSA. In addition, PGE2 significantly potentiated the ability of PAF-acether to elicit acute wheal (volume) and flare responses in human skin. The inflammatory properties of PAF-acether should not be considered in isolation since this phospholipid interacts synergistically with prostaglandins which are recognised as modulators of inflammation.