Analysis of histamine and N tau-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry

Current methods of quantitation of histamine and its major metabolite N tau-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [2H4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 430/434. For N tau-methylhistamine, after the addition of N tau-[2H3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C3F7CO2)2-N tau-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 497/500. The precision of the histamine assay is 3.1% and the accuracy is 95.5 +/- 2.5% while the precision of the N tau-methylhistamine assay is 1.9% and the accuracy is 106.8 +/- 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N tau-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N tau-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N tau-methylhistamine in plasma samples.     

Activation of plasma Hageman factor and kallikrein in ongoing allergic reactions in the skin

Ten atopic subjects, sensitive to intradermal injection of less than or equal to 10 protein nitrogen units of ragweed or grass pollen antigen, underwent paired antigen and buffer skin chamber incubation over the base of denuded skin blisters. The chamber fluids were sampled over a 6-hr period for histamine and activated Hageman factor and plasma kallikrein which were complexed to C1 inhibitor. In 9 of 10 subjects significantly (p less than 0.01) increased histamine levels (74 +/- 11 ng/ml vs 1.5 +/- 0.55 ng/ml) and kallikrein-C1 inhibitor complexes (2.15 +/- 0.78 ng/ml/hr vs 0.51 +/- 0.09 ng/ml/hr, p less than 0.25) were detected at antigen sites compared with buffer sites, respectively. Increased levels of activated Hageman factor (ng/ml/hr) were detected at antigen sites (1.35 +/- 0.60) compared with buffer sites (0.11 +/- 0.05), (p less than 0.01), in 8 of 10 subjects. Whereas peak levels of histamine were obtained after 1 hr of challenge, both Hageman factor and kallikrein activation, as assessed by complex formation, tended to peak later from the 2nd to the 5th hr. This represents the first demonstration that cutaneous IgE-mediated allergic responses are associated with local activation of the intrinsic plasma coagulation-kinin pathways.     

Protein kinase C (PKC) changes in human basophils. IgE-mediated activation is accompanied by an increase in total PKC activity

We have examined the changes in protein kinase C (PKC) which follow IgE-mediated activation of basophils. Exposure to 0.1 microgram/ml anti-IgE resulted in an increase in total cellular PKC (169 +/- 23% of control, histamine release (HR) = 33 +/- 7%, n = 12) which could be accounted for solely by the increase in membrane-associated PKC. These changes reached a maximum (280 +/- 48%) 1.0 min after challenge and declined to 190 +/- 38% after 5.0 min though histamine release was not complete until 5 to 10 min later. We found a good correlation between the increase in membrane-associated PKC and the eventual release of histamine (rs = 0.902). Donors whose basophils released less than 5% total histamine (n = 3, HR = 3 +/- 1%) showed a partial activation of PKC (173 +/- 18%) though much less than the remaining donors (increase in PKC = 346 +/- 59%, n = 9, HR = 43 +/- 7%). We observed no redistribution of cytosolic PKC at any time following exposure to anti-IgE. In contrast, 0.1 microgram/ml 2-O-tetradecanoyl-phorbol-13-acetate (HR = 36 +/- 3%, n = 3) promoted an increase in total cellular PKC, the loss of 31 +/- 4% of the cytosolic PKC and an 816 +/- 183% increase in membrane-associated PKC. Activation of PKC by anti-IgE was only partially dependent on extracellular calcium. In the absence of calcium, the increase in PKC was approximately 65% (n = 4) of that noted in the presence of 1mM calcium but these levels were sustained over much longer periods, failing to return to base line after 30 min. Higher than normal concentrations of calcium (5 to 10 mM) promoted rapid increases in PKC activity and accelerated the return to base line (back to prechallenge levels by 5 min). Suboptimal concentrations of anti-IgE (0.01 microgram/ml) attenuated the changes in membrane associated PKC and altered the kinetics of the response. The time required to reach maximum activity increased from 1.0 to 5.0 min with a corresponding decrease in the rate at which histamine was released. Higher concentrations of anti-IgE (1.0 microgram/ml) promoted a rapid increase in PKC (maximum increase in PKC = 501 +/- 59%, time = 0.5 min, HR = 28 +/- 2%) followed by an equally rapid return to base line levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Social suppression of ovarian cyclicity in captive and wild colonies of naked mole-rats, Heterocephalus glaber

To investigate the endocrine cause of reproductive suppression in nonbreeding female naked mole-rats, animals from 35 colonies were studied in captivity. Urinary and plasma progesterone concentrations were elevated in pregnant females (urine: 10.0-148.4 ng/mg Cr, 27 samples from 8 females; plasma: 3.6-30.0 ng/ml, 5 samples from 5 females; Days 21-40 of pregnancy) and cyclic breeding females (urine: 0.5-97.8 ng/mg Cr, 146 samples from 7 females; plasma: less than 1.0-35.4 ng/ml, 25 samples from 7 females). The latter group showed cyclic patterns of urinary progesterone, indicating a mean ovarian cycle length of 34.4 +/- 1.6 days (mean +/- s.e.m.) with a follicular phase of 6.0 +/- 0.6 days and a luteal phase of 27.5 +/- 1.3 days (19 cycles from 9 breeding females). In non-breeding females urinary and plasma progesterone values were undetectable (urine: less than 0.5 ng/mg Cr, 232 samples from 64 females; plasma: less than 1.0 ng/ml, 7 samples from 6 females). Breeding females had higher (P less than 0.001) plasma LH concentrations (3.0 +/- 0.2 mi.u./ml, 73 samples from 24 females) than did non-breeding females (1.6 +/- 0.1 mi.u./ml, 57 samples from 44 females). Urinary and plasma progesterone concentrations in non-breeding females from wild colonies situated near Mtito Andei, Kenya, were either below the assay sensitivity limit (urine: less than 0.5 ng/mg Cr, 11 females from 2 colonies; plasma: less than 1.0 ng/ml, 25 females from 4 colonies), or very low (plasma: 1.6 +/- 0.6 ng/ml, 15 females from 4 colonies). In captivity, non-breeding females removed from their colonies (i.e. the dominant breeding female) and either paired directly with a non-breeding male (N = 2), or removed and housed singly for 6 weeks before pairing with a non-breeding male (N = 5) may develop a perforate vagina for the first time in as little as 7 days. Urinary progesterone concentrations rose above 2.0 ng/mg Cr (indicative of a luteal phase) for the first time 8.0 +/- 1.9 days after being separated. These results suggest that ovulation is suppressed in subordinate non-breeding female naked mole-rats in captive and wild colonies, and show that plasma LH concentrations are significantly lower in these non-breeding females. This reproductive block in non-breeding females is readily reversible if the social factors suppressing reproduction are removed.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Central role of cyclooxygenase in the response of canine peripheral airways to antigen

We studied the effects of antigen aerosol challenge on the airways of the canine peripheral lung and examined the roles of cyclooxygenase products, histamine, and cholinergic activity in the responses. One-minute deliveries of 1:10,000 or 1:100,000 concentrations of Ascaris suum antigen aerosol through a wedged bronchoscope resulted in mean maximal increases in collateral system resistance (Rcs) of 415 and 177%, respectively, after 4-8 min. Repeated antigen challenge (1:100,000) resulted in significantly decreased responsiveness to antigen after the initial exposure (P less than 0.005). Bronchoalveolar lavage fluid obtained from the isolated, challenged segment had a significant increase in mean (+/- SE) prostaglandin D2 (PGD2) concentration vs. control (222.0 +/- 65.3 vs. 72.7 +/- 19.5 pg/ml; P less than 0.05); histamine concentrations were variable and not significantly different (4.1 +/- 2.6 vs. 1.2 +/- 0.2 ng/ml; P greater than 0.05). In nine experiments, cyclooxygenase inhibition significantly attenuated the antigen-induced increase in Rcs by 53.4% (P less than 0.001), and the concentration of PGD2 in lavage fluid was reduced by 96.0% (P less than 0.01). Blockade of histamine H1-receptors (n = 8) or cholinergic receptors (n = 7) did not significantly affect the airway response (P greater than 0.05). These data indicate that the canine peripheral lung responds in a dose-dependent manner to antigen aerosol challenge and exhibits characteristics of antigen tachyphylaxis. Results also suggest that cyclooxygenase products play a central role in the acute bronchoconstrictive response of the lung periphery.     

Further evidence for the usefulness of the salivary testosterone radioimmunoassay in the assessment of androgenicity in man in basal and stimulated conditions

Since correct assessment of testicular function and androgenic status in humans requires multiple sampling, a sensitive and accurate radioimmunoassay (RIA) of testosterone (T) was established for male and female saliva samples. This easily collected biological fluid, which contains nonprotein-bound T, may represent an attractive alternative or a complement to total plasma T assays. In saliva samples from 5 normal males, a clear circadian rhythm was observed, and morning concentrations (135 +/- 31 pg/ml) were significantly higher (p less than 0.02) than evening samples (85 +/- 23 pg/ml). In 11 normal females, morning saliva levels were 12.8 +/- 1.8 pg/ml. The levels of T in male saliva, in response to both exogenous T administration (100 mg i.m.) and HCG stimulation (2 X 2,000 IU i.m.), accurately reflected the changes observed in plasma T, and the magnitude of increase in T levels was clearly greater in saliva than in plasma samples during the intramuscular administration of the long-acting T preparation. In males, significant correlations were observed between salivary and plasma T concentrations in morning samples (r = 0.61, p less than 0.01), following HCG stimulation (r = 0.89, p less than 0.05) and during T administration (r = 0.87, p less than 0.05). In women, the correlation at 8 a.m. was also significant (r = 0.82, p less than 0.05).     

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.)

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.     

Distribution of bronchoconstrictor responses in isolated-perfused rat lung

We studied the effects of bronchoconstrictor stimuli administered selectively through isolated-perfused preparations of the bronchial and pulmonary circulations of 80 Sprague-Dawley rats. Dose-related contraction was elicited with infusion of acetylcholine (ACh), histamine, and serotonin (5-HT). Bolus infusion of 10(-5) mol ACh caused a 3.5-fold increase in pulmonary resistance (RL) after infusion into the pulmonary circulation (PC) and a 2.5-fold increase in the bronchial circulation (BC) (P less than 0.05 vs. control) that was blocked selectively in each circulation with atropine. Administration of 10(-5) mol 5-HT into the BC caused only a 45% increase in RL; the same dose of 5-HT caused a 5.1-fold increase in RL in the PC. A biphasic (increase at lower doses/decrease at higher doses) change in RL was elicited by histamine that was converted to dose-related constriction after H2-receptor blockade with cimetidine in both BC and PC. Response to exogenous ACh remained viable for greater than 5 h. Infusion of the mast cell degranulating agent, compound 48/80 (48/80), caused increase in RL that corresponded to quantitative recovery of histamine in the perfusates of both BC and PC. Histamine concentration in the perfusate increased from 47.2 +/- 31.8 (base line) to 624 +/- 60.1 ng/ml (2-fold increase in RL) in the BC and from 38.3 +/- 17.7 (base line) to 294.4 +/- 38.1 ng/ml (50% increase in RL) in the PC (P less than 0.001 vs. baseline concentration) after a 0.1-mg/ml dose of 48/80.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of ejaculation frequency on semen characteristics in chimpanzees (Pan troglodytes)

Semen samples were obtained by masturbation from 6 chimpanzees and the spontaneously liquefied fraction and the remaining coagulum were studied separately. When semen was collected once or twice a week, large intra-individual variations were observed for all measures. The liquefied fraction represented 26.5 +/- 3.2% (weighted mean +/- s.d.) of the total ejaculate but contained 51.3 +/- 3.8% of all emitted spermatozoa. Fructose concentration was higher in the coagulum than in the liquefied fraction (29.3 +/- 3.0 mumol/ml vs 12.0 +/- 2.7 mumol/ml, P less than 0.001) whereas acid phosphatase was less concentrated in the coagulum than in the liquefied fraction (3.5 +/- 0.3 x 10(3) IU/ml vs 13.0 +/- 0.9 x 10(3) IU/ml, P less than 0.001). L-Carnitine and citrate concentrations did not differ between the two fractions of the ejaculate. When semen collection was repeated every hour for 5 h, the ejaculate volume increased from 2.6 +/- 0.7 to 4.7 +/- 0.6 ml (P less than 0.001), whereas total sperm count decreased from 1278 +/- 872 x 10(6) to 587 +/- 329 x 10(6) (P less than 0.05) between the 1st and the 6th ejaculate. In the spontaneously liquefied fraction, the sperm count decreased from 984 to 369 x 10(6). The 6 successive ejaculates gave a total of 20.2 +/- 7.6 ml and 4278 +/- 2884 x 10(6) spermatozoa. The increase of the ejaculate volume was essentially due to an increase of the volume of the coagulum which closely correlated with total amount of fructose (from seminal vesicles) (r = 0.913, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

An in-vivo method for estimating inhibin production by adult rat testes

The concentrations of inhibin in samples of rat testicular venous and arterial blood and interstitial fluid were measured by an in-vitro bioassay using pituitary cells in culture in which the standard was an ovine testicular lymph preparation (assigned potency 1 unit/mg). Inhibin levels were undetectable (less than 2 U/ml) in both blood samples but reached a mean concentration of 120 +/- 7 U/ml in testicular interstitial fluid. After unilateral efferent duct ligation the rate of inhibin accumulation in seminiferous tubules was determined by the difference in the inhibin content of the ligated and unligated testes. Additionally, the rate of seminiferous tubule fluid production was obtained from the difference in weight between the ligated and non-ligated testes. In the 24 h after efferent duct ligation there were linear increases in inhibin (18.5 +/- 1.0 U/h) and in seminiferous tubule fluid production (26 +/- 1 microliter/h), but there were no changes in serum FSH and LH levels. Experimental induction of bilateral cryptorchidism led to a decrease in the inhibin content of the testis after 10 days. The rate of inhibin accumulation after efferent duct ligation declined more rapidly than the inhibin content, being significantly depressed in cryptorchid testes after 3 days, suggesting that this measurement is a more sensitive index of inhibin production than the determination of testicular inhibin content.     

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women.

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual period, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay. The patients receiving placebo had high PGF levels 135 +/- 27 ng/ml (Mean +/- S.E.) which were significnatly reduced by Ibuprofen to 24 +/- 5 ng/ml (P less than 0.001). The PGE concentrations decreased from 5 +/- 1 ng/ml to 2 +/- 1 ng/ml (P less than 0.05). Ibuprofen also reduced the menstrual pain significantly (P less than 0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.     

The characterization of radioimmunoassay for rat pancreatic polypeptide in serum.

A radioimmunoassay for the measurement of rat pancreatic polypeptide (RPP) in serum or plasma has been developed and characterized using a new guinea-pig anti-rat-PP antibody. The assay provides a high degree of sensitivity and lacks cross-reactivity (CR less than 0.01%) to neuropeptide Y and peptide YY. It also does not interact with PPs of other species or peptide hormones namely, amylin, glucagon, human insulin, human-PP, human-proinsulin, rat C-peptide and rat insulin. The assay employs synthetic rat PP as standards from concentrations of 21-2100 pg/ml (i.e., 5-500 pM) and produces a sensitivity limit of 19 pg/ml (4.5 pM) PP at +/- 3 S.D. The intra- and interassay % coefficient of variations are 6.4% and 5.9%, respectively. The % recovery of RPP added to rat serum samples ranges from 98% to 103%. Assay of serum volumes ranging from 25 microliters to 100 microliters does not significantly alter the expected RPP level. The migration patterns of rat serum PP and that of a synthetic RPP are identical by Sephadex G-50 chromatographic analysis. The mean values of fasting and a 2 h post-feeding plasma RPP levels in normal rats are 40 +/- 2 and 80 +/- 10 pg/ml (9.5 pM and 19.0 pM), respectively. Rat-PP release during insulin induced hypoglycemia in conscious rats rises from 38 +/- 5 pg/ml to 261 +/- 34 pg/ml (9.0 to 62.1 pM, P less than 0.005) by 30 min. Additionally, the antibody used in this study cross-reacts well with mouse-PP as determined by linear serum dilution curves, thus making it useful in the measurement of murine-PP. In conclusion, we have developed and validated a sensitive and specific rat-PP assay. This assay provides a new tool for the reliable measurement of PP in physiologic studies using rat and mouse animal models.     

Validation of the internal reference technique for microdialysis measurements of interstitial histamine in the rat

The internal reference technique (IRT) was compared with the no net flux method (NFM) as a microdialysis calibration technique for sampling of interstitial histamine in the rat. Microdialysis catheters (polyacrylonitrile, 50 kD cut off) were inserted in liver, muscle, subcutaneous tissue and in an induced adenocarcinoma. Estimated relative recovery with IRT ranged from 23+/-2% in liver to 30+/-3% in subcutaneous tissue with and without tumor (p<0.05). By using the NFM-technique we found similar recovery as compared to the IRT in all tissues studied. Interstitial histamine was up to 3-fold higher than the mean plasma histamine concentration (54+/-2 nmol/l). Subcutaneous tissue (177+/-39 nmol/l) and subcutaneous tumor (165+/-29 nmol/l) exhibited high histamine while liver (65+/-14 nmol/l) and liver tumor (75+/-7 nmol/l) had low interstitial histamine concentrations. In conclusion, the IRT was validated against the NFM as a rapid method for histamine measurements in situ in the rat.     

Recombinant human IL-1 alpha and -1 beta potentiate IgE-mediated histamine release from human basophils

In this study, we have explored the relationship between interleukins and human basophil activation. Previous studies by ourselves and others have found that recombinant human (rh) IL-3 causes histamine release. The ability to release histamine has also been claimed for IL-1 but we cannot confirm this. In experiments with the basophils of 29 donors (excluding one D2O responder), histamine release with 100 ng/ml rhIL-1 alpha was 1.3 +/- 1% (SEM), whereas with rhIL-1 beta, it was 0.8 +/- 1%. Both IL-1 alpha and -1 beta were also used at concentrations of 0.01 to 1000 ng/ml without causing release. Neither increasing the Ca2+ concentration nor adding D2O or cytochalasin B caused IL-1 alpha and -1 beta to become secretagogues. rhIL-1, however, did augment IgE-dependent histamine release. The enhancement was similar with both rhIL-1 alpha and -1 beta, i.e. they were dose-dependent between 0.1 and 3 ng/ml and reached a plateau from 3 to 100 ng/ml. At submaximal histamine release (less than 10%), there was enhancement of three IgE-dependent secretagogues: 125% with goat anti-human IgE (n = 7), 215% with Ag E (n = 10), and 260% with a histamine releasing factor (n = 7). Non-IgE-dependent stimuli (formyl-methionine-leucine-phenylalanine and the ionophore A23187, n = 10) were enhanced less than 5%. rhIL-1-enhancement persisted after cell washing (n = 10). rhIL-1 was active in preparations of 50 to 75% pure basophils in which mononuclear cells were reduced by greater than 95% (n = 4), and mAbH34 to IL-1 beta blocked the enhancement caused by that molecule. We postulate that basophils have an IL-1 receptor which, when occupied, upregulates the response to IgE-related signals. Thus, this work characterizes a second interaction between interleukins and the cells central to the allergic response.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Augmentation of respiratory mast cell secretion of histamine caused by vagus nerve stimulation during antigen challenge

We studied the effect of vagus nerve stimulation on the mast cell secretion of histamine after intraarterial (i.a.) administration of Ascaris suum antigen (AA) into the bronchial circulation of 10 randomly selected, natively allergic dogs in vivo. Respiratory mast cell response was measured as the arteriovenous difference (AVd) in histamine concentration across the bronchus. Plasma histamine concentration was determined simultaneously from right atrium, right ventricle, and femoral artery 60 and 15 sec before and 15, 30, 45, 60, 75, and 90 sec after i.a. injection of sham (Kreb-Henseleit) diluent, 1:100, and 1:30 concentrations of AA. The mean AVd in plasma histamine for five parasympathetically blocked animals (neural blockade with hexamethonium and beta-adrenergic blockade with propranolol) was 1.28 +/- 0.61 ng/ml (sham), 5.16 +/- 19.7 ng/ml (1:100 AA), and 36.6 +/- 11.1 ng/ml (1:30 AA). Substantial augmentation was obtained when AA was administered during parasympathetic stimulation in five other animals (beta-adrenergic blockade, no neural blockade), which was caused by continuous bilateral electrical stimulation of the vagus nerves. A mean AVd in plasma histamine of 110 +/- 27.6 ng/ml was obtained after 1:100 AA (p less than 0.001 vs parasympathetic blockade) and 166 +/- 32.4 ng/ml for 1:30 AA (p less than 0.001 vs parasympathetic blockade). Parasympathetic stimulation alone did not cause secretion of histamine. In contrast to the AVd response, parasympathetic stimulation did not augment nonrespiratory mast cell secretion after AA challenge. We conclude that vagus nerve stimulation augments secretion of histamine from respiratory mast cells during antigen challenge. We demonstrate that parasympathetic stimulation may potentiate the response to antigen challenge in central airways through augmented mast cell secretion of mediator.