Distribution of galanin-binding sites in the monkey and human telencephalon: preliminary observations

Summary Using X-ray film autoradiography the distribution of ¹²⁵I-galanin binding sites was studied in the forebrain of monkey and man. In the monkey a high density was found in all areas of the neocortex, especially layer 4, and in certain subfields in the hippocampal region. Also in the human brain high activity was seen in neocortex, mainly layer 6 and in hippocampal areas, as well as in amygdala, piriform cortex and hypothalamus. These results suggest that the 29-amino acid peptide galanin may be involved in the regulation of higher cortical functions in primates.     

Tachykinin-like immunoreactivity in the mammalian urinary bladder: correlation with the functions of the capsaicin-sensitive sensory nerves

The tachykinin-like immunoreactivity of the urinary bladder has been measured in various species by means of an antiserum (K12) having negligible cross-reactivity with substance P. The rank order for bladder content of tachykinin-like immunoreactivity was guinea-pig greater than mice greater than rat, similar to that found for substance P-like immunoreactivity. In all three species, both substance P- and tachykinin-like immunoreactivities were depleted by systemic capsaicin desensitization. The time course for depletion of substance P- and tachykinin-like immunoreactivities of the rat bladder following extrinsic denervation was almost superimposable. At reverse phase high pressure liquid chromatography, the major constituent of tachykinin-like immunoreactivity of the rat bladder co-eluted with neurokinin A. In vitro, the contractile response of the rat bladder to capsaicin (1 microM) was directly proportional to bladder tachykinin-like immunoreactivity while the response to field stimulation was not. In vivo, the volume threshold for reflex micturition was inversely proportional to bladder tachykinin-like immunoreactivity while amplitude of micturition contraction was not. Similar correlations were found in a previous study for substance P-like immunoreactivity. The contractile response to capsaicin or neurokinin A of the rat isolated bladder were significantly reduced by incubation with phenoxybenzamine at a concentration reported to produce a selective alkylation of neurokinin-2 receptors, while the response to substance P or KCl was unaffected. These findings indicate that multiple neurokinins co-exist in those bladder sensory nerves which are capsaicin-sensitive in adult rats. Both substance P- and tachykinin-like immunoreactivities in the rat bladder appear to be good functional markers of the sensory and "efferent" functions mediated by capsaicin-sensitive nerves, consistent with the hypothesis of a transmitter role for the corresponding peptides.     

Neurotensin-like Peptides in the CNS of Lampreys: Chromatographic Characterization and Immunohistochemical Localization with Reference to Aminergic Markers

Neurotensin (NT)-like peptides in the CNS of the lamprey Lampetra fluviatilis were studied by radioimmunoassay (C-terminal specific NT antiserum), reverse-phase HPLC and immunohistochemistry. Multiple peaks of NT-immunoreactive (-ir) material were observed upon HPLC, of which a major peak eluted in the position of bovine NT. Immunofluorescence histochemistry showed that a monoclonal antibody recognizing the N-terminal (1 - 11) fragment of NT, as well as two polyclonal NT antisera labelled a large number of cell bodies in the periventricular area of hypothalamus, including the postinfundibular commissural nucleus and the ventral and dorsal hypothalamic nuclei. Additional groups of NT-ir cells were observed in the preoptic nucleus, the postoptic commissural nucleus, the mesencephalic tegmentum (L.fluviatilis), and in the spinal cord (L.fluviatilis and Ichtyomyzon unicuspis). Dense NT-ir fibre plexuses were present in the caudal hypothalamus, corpus striatum, ventral mesencephalon, and in the dorsal horn and lateral margin of the spinal cord. At the ultrastructural level the lateral spinal margin showed NT-ir terminal structures, which in most cases were not associated with synaptic specializations, although occasional synaptic contacts with unlabelled elements were found. The relation between NT-ir and monoamine-containing cells was examined with immunofluorescence double-staining, using antisera to tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and histamine respectively. In the periventricular nuclei of hypothalamus numerous TH-, 5-HT-, as well as histamine-ir cells were located in close association with NT-ir cells, but none of the aminergic markers could be detected within NT-ir neurons. The chemical properties as well as the anatomical distribution of lamprey NT-like peptides show several similarities with those present in mammals, suggesting that NT-containing neuronal systems in the CNS developed early in vertebrate phylogeny.     

Glutamine supplementation does not prevent small bowel mucosal atrophy after total parenteral nutrition in the rat

Glutamine supplementation to non-lipid parenteral nutrition has been demonstrated to attenuate villus atrophy and increase mucosal DNA content in the rat. This study was performed in order to determine the effects of glutamine supplementation to a balanced TPN mixture (including lipids) on epithelial cell kinetics using autoradiography. Male Sprague-Dawley rats were used. Group 1 (control) received food and an intravenous saline infusion. Group 2 received an intravenous TPN mixture including lipids but without glutamine. The same TPN mixture, glutamine replacing an isonitrogenous amount of non-essential amino acids, was given to Group 3. Animals were fed for 7 days, whereafter blood and intestinal samples were taken 1 h after injection of tritiated thymidine. Microscopy of specimens from proximal jejunum revealed a significant reduction in the number of cells in crypts and villi in both TPN groups (2 and 3) compared to orally fed animals (p < 0.001). Epithelial cell numbers were not significantly different in Group 2 and 3. Similarly, the labelling index (number of labelled cells/number of crypt cells) was not affected by glutamine administration. In plasma, glucagon concentrations in Group 2 (TPN without glutamine) seemed to decrease compared to Group 1 and 3 (p = 0.06). In this study, glutamine supplementation did not affect apithelial atrophy or cell proliferation. It is concluded, that the effects of glutamine on mucosal atrophy and renewal in jejunum may depend on the composition of the TPN mixture supplied during parenteral feeding.     

Radioimmunoassay for rat galanin: immunochemical and chromatographic characterization of immunoreactivity in tissue extracts

Galanin is a regulatory peptide with wide distribution in the central and peripheral nervous system and with numerous biological effects. Several radioimmunoassays based on antisera raised against porcine galanin have been used to measure immunoreactivity in rat tissues. However, considerable lack of parallelism has been observed between the porcine standard and rat tissue extracts, which may decrease the reliability of the quantitative data. The purpose of the present study was therefore to raise antibodies against rat galanin and establish a competitive radioimmunoassay for rat galanin. Two antisera, RatGal4 and RatGal5, were characterized in detail. The homogeneity of the immunoreactive material from several tissues was also investigated with column chromatography. At reverse-phase high-pressure liquid chromatography more than 95% of the immunoreactive material from rat CNS eluted as a single peak in the position of synthetic rat galanin, whereas almost half of the immunoreactive material from the intestine eluted in positions different from the synthetic peptide. Extracts of rat brains as well as jejunum diluted in parallel with the standard curve for both antisera. We conclude that measurements of rat galanin based on these antisera are therefore more reliable than those based on antisera raised against porcine galanin.     

Stress at three‐month immunization: Parents’ and infants’ salivary cortisol response in relation to the use of pacifier and oral glucose

The aims of the present study were to investigate (1) whether the salivary cortisol response could be dampened during a routine three‐month immunization if the infant received sweet‐tasting solution in combination with a pacifier and (2) stress experienced by parents during immunization of the infant. Ninety‐eight infants were included into one of four intervention groups: ‘glucose and pacifier’, ‘water and pacifier’, ‘glucose’, or ‘water’. Saliva was collected before and 30min after the immunization. Infants’ crying‐time and parents’ self‐reported stress (VAS) were measured before and after immunization. Infants in the ‘pacifier and glucose’ group had a significantly smaller change in salivary cortisol than infants in the other groups (F_3,72=3.1, p<0.05). In the ‘glucose and pacifier’ group the median salivary cortisol levels decreased 33% after the immunization. In the ‘water and pacifier’, ‘glucose’, and ‘water’ group median cortisol increased with 50%, 42%, and 8%, respectively. No significant differences in crying‐time were observed between the intervention groups. If the infant cried before the immunization, the crying‐time during the immunization was longer (p<0.01) and cortisol increased more (p<0.05). Median cortisol levels for parents decreased after the immunization (p<0.01). Median VAS increased 50% (p<0.0001) after immunization. First time parents rated higher stress on VAS before immunization (p<0.01). Parents’ change in cortisol and VAS were significantly related to infants’ crying time. In conclusion, the combination of oral glucose and pacifier dampen infants’ salivary cortisol in response to the three‐month immunization.     

Methods for long-term 17β-estradiol administration to mice

Rodent models constitute a cornerstone in the elucidation of the effects and biological mechanisms of 17β-estradiol. However, a thorough assessment of the methods for long-term administration of 17β-estradiol to mice is lacking. The fact that 17β-estradiol has been demonstrated to exert different effects depending on dose emphasizes the need for validated administration regimens. Therefore, 169 female C57BL/6 mice were ovariectomized and administered 17β-estradiol using one of the two commonly used subcutaneous methods; slow-release pellets (0.18 mg, 60-day release pellets; 0.72 mg, 90-day release pellets) and silastic capsules (with/without convalescence period, silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with a 14 mm column of 36 μg 17β-estradiol/mL sesame oil), or a novel peroral method (56 μg 17β-estradiol/day/kg body weight in the hazelnut cream Nutella). Forty animals were used as ovariectomized and intact controls. Serum samples were obtained weekly for five weeks and 17β-estradiol concentrations were measured using radioimmunoassay. The peroral method resulted in steady concentrations within--except on one occasion--the physiological range and the silastic capsules produced predominantly physiological concentrations, although exceeding the range by maximum a factor three during the first three weeks. The 0.18 mg pellet yielded initial concentrations an order of magnitude higher than the physiological range, which then decreased drastically, and the 0.72 mg pellet produced between 18 and 40 times higher concentrations than the physiological range during the entire experiment. The peroral method and silastic capsules described in this article constitute reliable modes of administration of 17β-estradiol, superior to the widely used commercial pellets.     

Tachykinins Influence Interdigestive Rhythm and Contractile Strength of Human Small Intestine

The effect of the putative entericneurotransmitters neurokinin A and substance P wereinvestigated on human small intestinal motility. Eitherneurokinin A, at doses of 6-25 pmol/kg/min, or substanceP at doses of 1- 6 pmol/kg/min were administeredintravenously to healthy volunteers over 4 hr.Neurokinin A dose-dependently increased the fraction ofphase II of the migrating motor complex, contraction frequency, motility index, and amplitude ofcontractions. At the highest dose, neurokinin A induceda phase II-like pattern, disrupting the migratingmyoelectric complex. Substance P dose-dependentlyincreased phase II of the migrating motor complex. Thecontraction frequency increased slightly at the highestdose, but neither motility index nor contractionamplitude changed. It is concluded that neurokinin A and substance P stimulate small intestinalmotility in man, and it can be speculated that they playa role in the control of human small intestinalmotility.     

Influence of angiotensin converting enzyme inhibition on relation of atrial natriuretic peptide concentration to atrial pressure in heart failure.

OBJECTIVE--To examine the influence of the duration of follow up on the values of heart rate variability (HRV) and the left ventricular ejection fraction (LVEF) for predicting mortality after infarction. BACKGROUND--HRV is an index of autonomic balance that identifies patients at a high risk of arrhythmic events. The index is most depressed during the first few weeks after myocardial infarction whereas left ventricular function tends to deteriorate with time. HYPOTHESIS--The value of depressed HRV measured before discharge from hospital for predicting mortality after infarction should decline with time. METHODS--The HRV and the LVEF were assessed in 433 survivors of a first acute myocardial infarction: HRV < 20 units and LVEF < 40% were taken as cut off points. Kaplan-Meier survival functions for total cardiac mortality and sudden cardiac death were calculated for the whole five year follow up period and for different intervening periods. RESULTS--During follow up of four weeks to five years there were 46 (10.6%) deaths and 15 (3.5%) patients died suddenly. Within the whole follow up period, HRV < 20 units and LVEF < 40% were both strongly associated with total cardiac mortality (p < 0.0001), but HRV was an independent predictor of total cardiac mortality only during the first six months of follow up. There were no deaths predicted by HRV < 20 units after the first year of follow up whereas LVEF < 40% had a sensitivity of 43% and a positive predictive accuracy of 9% for predicting death during this period. HRV < 20 units was better than LVEF < 40% in predicting sudden deaths during the first year of follow up but was an independent predictor only of those sudden deaths occurring within six months of infarction. CONCLUSIONS--The duration of follow up affects the prediction of sudden death and total cardiac mortality from HRV. Reduced HRV as measured before discharge from hospital does not seem to retain independent prognostic value after six months of follow up. These findings have potential implications for the serial evaluation of HRV and for the prevention of sudden death after myocardial infarction.