Presence of isolated tumour cells in mesenteric lymph nodes predicts poor prognosis in patients with stage II colon cancer.

AIM: Most patients with stage I and stage II colon adenocarcinomas do not have disseminated disease, and the group is not offered adjuvant therapy. However, more than 30% of stage II colon adenocarcinoma patients get metastases to remote organs. Thus, it is important to identify patients in this group at risk of disease relapse. PATIENTS AND METHODS: We have examined the prognostic value of isolated tumour cells (ITC) in mesenteric lymph nodes in a consecutive series of 156 colon carcinoma patients with stage II disease. Immunohistochemistry, using antibodies to cytokeratins, and morphology were used to identify presence of ITC. RESULTS: ITC were detected in 59 (37.8%) patients. Presence of ITC in mesenteric lymph nodes was independently associated with reduced relative survival both in univariate (p=0.0199) and in a multivariate analysis (p=0.041). CONCLUSION: The results strongly suggest that presence of ITC in mesenteric lymph nodes is associated with reduced relative survival in colon carcinoma patients stage II, and that detection of ITC may be important in treatment of these patients.

5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism.

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products. Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised. Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium. There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase. Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting [3H]uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis. Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU. In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations. The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine. These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

Ortho-substituted polychlorinated biphenyls activate respiratory burst measured as luminol-amplified chemoluminescence in human granulocytes.

The effect of polychlorinated biphenyls (PCBs) on the activation of respiratory burst measured as luminol-amplified chemoluminescence in human granulocytes is elucidated here. Chemoluminescence was stimulated in a concentration-dependent manner (ED50 approximately 10 microM) by ortho-substituted PCB congeners, while meta- and para-substituted congeners had no significant effect. Two ortho-substituted PCB congeners were chosen for the mechanistic studies, namely 2,2',4,4'-TeCB and 2,2'-DCB, since they have been used in previous studies by others. In the absence of extracellular calcium, the respiratory burst in response to 2,2'-DCB and 2,2',4,4'-TeCB was reduced by 63% and 82%, respectively. Bisindolylmaleimide, which inhibits protein kinase C, reduced activated chemoluminescence by 2,2'-DCB, 2,2',4,4'-TeCB, N-formyl-methionyl-leucyl-phenylalanine, and phorbol 12-myristate 13-acetate. Neomycin, which inhibits phospholipase C, had a slight, but significant, effect on the 2,2',4,4'-TeCB-activated chemoluminescence but had a more pronounced effect on the 2,2'-DCB-activated chemoluminescence. 2,2'-DCB and 2,2',4,4'-TeCB significantly increased phospholipase D (PLD) activity measured as the amount of 14C-phosphatidylbutanol formed. Ethanol (1%), a phospholipase D modulator, reduced the response to 2,2'-DCB and 2,2',4,4'-TeCB by 72% and 75%, respectively. Furthermore, wortmannin (25 nM), a phosphatidylinositol 3-kinase, and genistein, a more unspecific tyrosine kinase inhibitor, reduced chemoluminescence in response to PCB. In conclusion, our results indicate that PCB-activated chemoluminescence is dependent on the Ca(2+)-dependent phospholipase D or phospholipase C, phosphatidylinositol 3-kinase, and protein kinase C activation prior to activation of the NADPH oxidase. Defects in neutrophhil functions upon exposure to PCB may render a greater susceptibility in the host to invading microorganisms or evoke inappropriate inflammatory responses leading to tissue injury.

Glucocorticoid and ACTH regulation of rat peritoneal phagocyte chemiluminescence and nitric oxide production in culture.

In order to study the adrenocortical regulation of monocyte/macrophage functions further, leucocytes from the rat peritoneum were incubated in vitro with glucocorticoid concentrations up to 10 mumol L-1 and with adrenocorticotropic hormone (ACTH) up to 100 micrograms mL-1. The monocyte/macrophage production of reactive oxygen molecules was measured by luminol amplified chemiluminescence, and the production of nitric oxide (NO) was measured as nitrite (NO2-). Dexamethasone in vitro in nanomolar concentrations inhibited monocyte/macrophage chemiluminescence and also nitric oxide production; the potency was dexamethasone > methylprodnisolone > prednisolone. ACTH enhanced both activated chemiluminescence and endotoxin-induced nitric oxide production, but only at concentrations about 20-100 micrograms mL-1, and there was no significant effect of physiological concentrations. In summary, the results of the present study further confirm and substantiate that glucocorticoids in low pharmacological concentrations have a general inhibitory effect on monocyte/ macrophage production of reactive oxygen molecules through the specific glucocorticoid receptors, while the stimulatory effect of ACTH is only observed by very high, non-physiological concentrations. Furthermore, since low concentrations of glucocorticoids inhibited the production of these reactive oxygen molecules in vitro, indirect mechanisms involving hormones and other elements outside the immune system are not essential for the effect of glucocorticoids on monocytes/macrophages.

Peritoneal leucocytes from spontaneously hypertensive rats have reduced chemiluminescence response and lowered sensitivity to dexamethasone in vivo.

The genetically altered hypothalamo-pituitary-adrenocortical axis in the spontaneously hypertensive rat (SHR) suggests altered phagocyte function in this strain. We therefore compared luminol-amplified chemiluminescence in peritoneal leucocytes from 10- to 12-week-old SHRs and age-matched Wistar-Kyoto rats (WKYs) activated by serum-opsonized zymosan particles (SOZ), N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). While the number of peritoneal monocytes/macrophages was increased by 49% in SHRs relative to WKYs, activator-induced chemiluminescence per cell in SHRs was only 14-42% of that in WKYs. FMLP responses were especially low in SHRs. Treatment of rats with dexamethasone in the drinking water for 48 h prior to ex vivo experiments reduced chemiluminescence dose-dependently in WKYs as well as in SHRs. ED50 of dexamethasone in SHRs was, however, increased compared to WKYs, indicating lowered sensitivity to dexamethasone in SHRs. No evidence was found of strain differences in differential distribution of peritoneal cells or in pharmacokinetics of dexamethasone. Plasma ACTH levels were significantly higher in SHRs than in WKYs, while basal plasma corticosterone concentrations in SHRs and WKYs were not significantly different. The results suggest that production of reactive oxygen compounds by peritoneal mononuclear phagocytes is reduced in SHRs compared with WKYs, and that the phagocyte respiratory burst is modulated differently by endogenous glucocorticoids in the two strains. We propose that reduced activity of the phagocyte NADPH oxidase-myeloperoxidase system is a major contributory cause of the altered chemiluminescence responses in SHRs. The data indicate that species differences may also be present at earlier steps in the signal transduction pathways activated by SOZ, fMLP and PMA.

The effect of strenuous exercise, calorie deficiency and sleep deprivation on white blood cells, plasma immunoglobulins and cytokines.

Moderate exercise appears to stimulate the immune system, but there is good evidence that intense exercise can cause immune deficiency. In the present study the authors examined the effect of continuous physical exercise (35% of VO2 max), calorie deficiency and sleep deprivation on the immune system of young men participating in a 5-7 days military training course. There was a two-three fold increase of neutrophils from day 1, the values remained high and decreased slightly at the end of the course. Monocyte counts also increased with a pattern similar to that of neutrophils. Eosinophils decreased to 30% of control and lymphocyte numbers decreased by 30-40%. All the major subgroups (CD4 T cells, CD8 T cells, B cells, NK cells) were reduced. Neutrophil function, as tested by measuring chemotaxis, was significantly stimulated during the first days of the course, in particular in the group with the lowest calorie intake. The mitogenic response of lymphocytes to PHA and Con A was variable, ranging from stimulation during one course to no effect in another course. Serum levels of immunoglobulins decreased significantly during the course. IgG was reduced by 6-7%, IgA by 10-20% and IgM by 20-35%. The authors found no changes of interleukin 1, 2 and 4 during the course, but a (12-20%) reduction (P less than 0.01) of interleukin 6, and an increase (P less than 0.01) of granulocyte-macrophage colony stimulating factor. Altogether the results from the ranger course present a mixed-up picture. The non-specific phagocyte-related immunity was enhanced. On the other hand, the data indicate that even a moderate physical activity, around the clock, caused significant suppression of a number of parameters reflecting the status of the specific, lymphocyte-related immunity. It is noteworthy, however, that there was no significantly increased infection rate during the course or in the first 4-5 weeks thereafter.

Granulocyte chemiluminescence response to serum opsonized zymosan particles ex vivo during long-term strenuous exercise, energy and sleep deprivation in humans.

The chemiluminescence response of granulocytes to serum opsonized zymosan particles (SOZ) ex vivo was investigated during two ranger training courses lasting 7 days with continuous moderate physical activities corresponding to about 32% of maximal oxygen uptake or 35000 kJ.24 h-1, with energy deficiency (energy supply 0-4000 kJ.24 h-1), and less than 3-h sleep during the 7 days. Significant granulocytosis in combination with a lymphopenia in peripheral blood was observed during the whole course. A priming of the granulocytes for accentuated chemiluminescence response to SOZ was observed during the first days of the course with a maximal increase on day 3 in course A (+35% of control response) and on day 1 in course B (+12%). Thereafter, reduced responses to SOZ compared to control values (-28% and -21% in course A and B) were observed. In course A, a group (n = 8) receiving 5000 kJ.24 h-1 of additional energy, showed a more pronounced priming (maximum +57% versus +21% of control response) during the first days. In course B, all the cadets had 3 h of organised rest/sleep on day 5, and a second priming of the chemiluminescence response was observed on the subsequent 2 days. These data indicated that moderate, continuous, predominantly aerobic physical activities for 1-3 days around the clock primed the production of reactive oxygen species in granulocytes. This priming may be beneficial for, for example, host defence against micro-organisms, but may also contribute to inflammatory damage to normal tissues such as muscle, tendons and joints during exercise. However, when the moderate exercise continued for several more days, a down-modulation of the granulocyte response was observed. The findings of this study further support the possibility that moderate physical activity stimulates immunity, while more extreme duration of the same activities may result in a down-modulation of non-specific (and specific) immunity.

Dexamethasone and methylprednisolone affect rat peritoneal phagocyte chemiluminescence after administration in vivo.

Production of reactive oxygen compounds by peritoneal monocytes/macrophages was studied in rats exposed to dexamethasone or methylprednisolone in the drinking water. Luminol-amplified chemiluminescence was measured in preparations of peritoneal leukocytes activated ex vivo by serum opsonized zymosan, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). After dexamethasone administration for 1 day (approximately 0.13 mg/kg per 24 h) a significant reduction in chemiluminescence was found in cells stimulated with serum opsonized zymosan, while responses to fMLP and PMA stimulation were significantly reduced after 2 days. The maximal inhibition obtained after 5-8 days of dexamethasone administration (plasma levels < 5 nM) was 92.0 +/- 1.2%, 87.6 +/- 0.2% and 84.5 +/- 3.1% in cells stimulated with serum opsonized zymosan, fMLP and PMA, respectively. Administration of dexamethasone or methylprednisolone for 48 h gave a dose-dependent reduction of chemiluminescence. ED50 values of dexamethasone were estimated at 0.06-0.15 mg/kg for the different stimulators (plasma concentrations 5-10 nM). Estimated ED50 values for methylprednisolone were 35-36 mg/kg. Since the percentage of mononuclear phagocytes in the peritoneal cell population did not change significantly with dose or time of dexamethasone exposure, this study indicates that glucocorticoids have a depressive effect on the monocyte/macrophage 'respiratory burst' in vivo. The results are consistent with the hypothesis that these effects are mediated by glucocorticoid receptors. Although the pathway activated by serum opsonized zymosan was more rapidly inhibited than the fMLP- and PMA-activated pathways, the responses induced by the different stimulators were similarly affected, suggesting a modulation of common components in the activation pathways, possibly protein kinase C or the NADPH-oxidase complex, after administration of low pharmacological doses of glucocorticoids in vivo.

Effect of in vivo corticosterone and acute food deprivation on rat resident peritoneal cell chemiluminescence after activation ex vivo.

Adrenoglucocorticoid regulation of rat peritoneal monocyte/macrophage function was studied by exposing rats to corticosterone (CS) in the drinking water, and to fast (48 h). Production of reactive oxygen metabolites was measured by luminol amplified chemiluminescence (CL) in preparations of peritoneal cells activated by serum treated zymosan (STZ). Administration of CS which led to an increase in plasma CS from 31 (controls) to 46 ng mL-1, reduced CL (per cell) by 31%. Fast, which did not change plasma CS or ACTH, also had an inhibitory effect on CL (-25%), while the combination of CS administration and fast strongly inhibited the CL (-89%), indicating that plasma CS and fast reduced CL in a synergistic way. Similar effects on cell number were observed: CS-administration, fast and the combination reduced macrophage numbers (-13, -19.7 and -55%), while no significant effect was observed on the number of monocytes. The effect of adrenalectomy (adx) was studied in another series of experiments; adx induced no significant change in peritoneal leucocyte number or composition, while cells from adx animals had significantly higher chemiluminescence reaction than cells from sham operated animals. CS substitution in adx animals reduced CL by 30% while sham operated animals had 49% lower CL in adx. The data from adx animals also suggest that endogenous levels of CS are inhibitory for CL, but the results are not conclusive for the effect of very low doses of CS since other mechanisms than elimination of CS could prime the chemiluminescence reaction after adx. In conclusion, a moderate elevation of CS after systemic administration in vivo reduced the total number of mononuclear phagocytes in rat peritoneum, reduced the relative number of macrophages compared with monocytes, and suppressed the function of monocytes/macrophages by reducing the production of reactive oxygen molecules in activated cells. Furthermore, the effect of corticosterone was also dependent on the physiological situation, since the effects of fast and corticosterone were synergistic.

Adrenaline stimulated cyclic adenosine monophosphate response in leucocytes is reduced after prolonged physical activity combined with sleep and energy deprivation.

The mechanism for adrenergic desensitisation during physical stress was studied by measuring [125I] cyanopindolol ([125I]CYP) binding sites and the adrenaline stimulated cyclic adenosine monophosphate (cAMP) responses in peripheral blood leucocytes from ten male cadets during a 5-day military training course. The cadets had physical activities around the clock corresponding to a daily energy consumption of about 40,000 kJ but with an intake of only 2,000 kJ, and only 1-3 h of sleep in the 5 days. During the course, the maximal cAMP response to adrenaline stimulation was reduced to about 45% in granulocytes and to 52% in mononuclear cells, and the half maximal response was obtained only at 5-10 times higher adrenaline concentrations than in the control experiment. The binding sites for [125I]-CYP in mononuclear cells increased during the course. However, [125I]-CYP measured not only surface receptors but also intracellular receptors and might even have represented other binding sites. In conclusion, this study showed that decreased cAMP response to adrenergic stimulation would seem to be one of the mechanisms behind adrenergic desensitisation during stress.

Effect of norethisterone and levonorgestrel in low-dose multiphasic oral contraceptives on serum lipids.

In a parallel, multicenter study in Norway and Finland involving a total of 196 healthy women (mean age 22.4 years, range 18-30), the effects on serum lipids and lipoproteins of two multiphasic oral contraceptives containing ethinyl estradiol (EE) but different progestins were examined. One formulation contained EE 35 micrograms and norethisterone (NET) 0.5 mg on days 1-7 and days 17-21 and elevated NET 1.0 mg during the midphase (days 8-16). The other formulation contained EE 30 micrograms on days 1-6 and days 12-21 and 40 micrograms on days 7-11 and phased levonorgestrel (LGN): 50 micrograms (days 1-6), 75 micrograms (days 7-11) and 125 micrograms (days 12-21). Both formulations induced significant elevation of total cholesterol (6.7 and 4.1%), Apo B (8.1 and 7.0%) as well as HDL (6.4 and 3.7%) for the EE/NET and EE/LGN formulation respectively. Mean serum levels of triglycerides were significantly elevated (58 and 47%). However, all mean serum lipid and lipoprotein values remained within the normal range, and no change in the calculated cholesterol ratio (HDL/total cholesterol) nor lipoprotein ratio (HDL/(HDL+LDL)) was observed. No significant difference between the formulations could be detected with respect to the effect on serum lipids and lipoproteins measured. The change in total cholesterol was smaller than reported in many studies of monophasic preparations. Taken together, these data suggest that only small alterations in lipid metabolism are elicited by these oral contraceptives.

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency.

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 microM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

Vasoactive intestinal peptide inhibits the respiratory burst in human monocytes by a cyclic AMP-mediated mechanism.

The neuropeptide vasoactive intestinal peptide (VIP) was shown to inhibit the production of reactive oxygen compounds (respiratory burst) in monocytes activated by serum opsonized zymosan. Reactive oxygen compounds are of importance for host defence against micro-organisms and cancer, but normal tissues are also susceptible to damage from these reactive substances. Maximum inhibition of respiratory burst was 40% by 0.1 microM VIP (ID100), while ID50 for the VIP effect was 0.36 nM VIP. PHM-27, closely related to VIP on the basis of the amino acid sequence, inhibited the respiratory burst with much lower potency (ID50 = 60 nM, ID100 = 1 microM). Secretin, related to VIP and PHM-27, produced no effect on the respiratory burst in monocytes. VIP was also shown to stimulate the cyclic AMP production in monocytes in a dose dependent manner. IBMX and forskolin, as well as the cyclic AMP analogue butyryl cyclic AMP were shown to produce an inhibition of the respiratory burst. In conclusion, this study showed that VIP inhibited the respiratory burst in monocytes by a cyclic AMP-mediated mechanism, and serves to establish still another role for VIP as a mediator in the neuro-immune axis.

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes.

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 microM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. Bmax of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 microM VIP. No significant change was observed in receptor affinity, in Bmax of the low-affinity receptor, in ED50, or in ED100 of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 microM, 3 h) caused 24% reduction in [125I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 microM, 3 h).

Receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes are upregulated during prolonged strain and energy deficiency.

VIP receptors on blood mononuclear leucocytes and plasma VIP concentrations were studied during a ranger training course lasting for five days with almost continuous physical activity, and energy deficiency. The maximum binding capacity (Bmax) for the high affinity receptor increased (p less than 0.0005) from 0.71 (SEM = 0.11, N = 10) fmol/million cells to a maximum of 7.33 (SEM = 1.0) fmol/million cells on Day 4. There was no significant change in the dissociation constant (Kd) for the high affinity receptor, and no effect on Kd nor Bmax for the low affinity VIP receptor was detected. Plasma VIP concentration increased (p less than 0.0005) from 8.8 pmol/l (SEM = 0.6) to a maximum of 23.4 (SEM = 1.9) on the second day of the course. However, the highest plasma concentrations were about one order of magnitude lower than the dissociation constant (Kd) for the high affinity VIP receptor on the mononuclear leucocytes. These data indicate that heterologous upregulation of the high affinity VIP receptor on mononuclear blood cells takes place during combined strenuous physical exercise, and calorie deficiency.

Binding and degradation of vasoactive intestinal peptide in prolactin-producing cultured rat pituitary tumour cells (GH4C1).

Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) secretion from cultured rat pituitary cells (GH cells) (Bjøro et al. 1984). This study demonstrates the presence of specific receptors for 125I-VIP on the GH4C1 cells. Specific binding was rapid and biphasic giving a transient plateau lasting from 7 to 30 min. Thereafter specific binding declined to about one-third after 90 min. This coincided with enhanced degradation of 125I-VIP. The degradation was mainly cell-mediated and only partly receptor dependent. Trichloroacetic acid precipitation and absorption chromatography indicated that the degradation products were either 125I- and/or small labelled peptide fragments. Bioassay, RIA and rebinding studies also demonstrated degradation of VIP. Pretreatment of GH4C1 cells with trypsin decreased the rate of degradation of 125I-VIP, but also reduced the amount of specific binding. Scatchard analysis of binding data indicated the existence of two independent classes of receptors, one with Kd = 2.2 nM and Bmax = 15 fmol per 10(6) cells and another with Kd = 180 nM and Bmax = 550 fmol per 10(6) cells. The IC50 for VIP, PHI and secretin were 4, 5 and 500 nM, respectively. We conclude that the high affinity receptor is the most probable mediator of VIP on PRL secretion. The effect of VIP and PHI on PRL secretion in GH4C1 cells is mediated through one common receptor.

Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites.

Vasoactive intestinal polypeptide (VIP) interaction with a 94% pure preparation of monocytes isolated from human peripheral blood was studied by direct binding technique using 3-[125I]tyrosyl-VIP as a tracer ligand. Scatchard analysis of binding data was compatible with two classes of binding sites, one with Kd = 0.25 nM and maximal binding capacity of 16 fmol/10(6) cells, and another one with Kd = 25 nM and maximal binding capacity of 180 fmol/10(6) cells. The binding was time-, temperature-, and pH-dependent and was saturable, reversible, and specific. This study has demonstrated that human monocytes have high affinity/low capacity as well as low affinity/high capacity binding sites for VIP. No specific VIP binding was found in pure preparations of human granulocytes, platelets or erythrocytes.