Hepatic bile formation in the rat

While changes in gastric, pancreatic, and intestinal secretion in response to more recently identified gastrointestinal peptides have been characterized, there has been less investigation into effects of these hormones on hepatic bile production. The isolated perfused rat liver model has been used to examine effects of vasoactive intestinal peptide (VIP), somatostatin, bombesin, and thyrotropin-releasing hormone (TRH) on bile flow and bile acid transport. No changes were seen following bolus administration of bombesin (3×10^–8–1.5×10^–6 M) or TRH (3×10^–7–3×10^–6 M), while somatostatin (6×10^–6 M) produced a small decrease in bile flow without any change in bile acid output. VIP (3×10^–7 M) caused a highly significant increase in both volume of bile flow (0.85±0.8 to 1.11±0.09 l/min/g liver,P<0.001) and bile acid output (31.6±1.5 to 43.2±1.7 nmol/min/g liver,P<0.001). Elimination of Ca²⁺ from liver perfusate did not prevent VIP-induced increases in bile flow and bile acid output, and no synergistic effect of concomitant theophylline administration was observed. While effects of VIP on bile flow appear to be due to alterations in hepatic transport of bile acids, the exact mechanism(s) producing these changes remains to be elucidated.This work was supported by National Institutes of Health grant AM-32208 and the Research Service of the Veterans Administration.     

Synergistic action of melatonin and vasoactive intestinal peptide in stimulating cyclic AMP production in human lymphocytes

In the present study we investigated the synergistic effect of melatonin and vasoactive intestinal peptide (VIP) on cyclic AMP production in human blood lymphocytes. As shown by our group previously, VIP alone behaved as a potent activator of cyclic AMP production in human lymphocytes. On the other hand, melatonin alone did not affect the intracellular levels of cyclic nucleotide at any time or dose studied. However, when cells were incubated with melatonin plus VIP, melatonin potentiated the effect of the peptide. This effect can be observed in the presence of physiological doses of both melatonin (10-100 pM) and VIP (1-100 pM). The effect is specific for VIP because with other peptides belonging to the secretin-VIP family the effect was not observed. Results suggest that melatonin, in conjunction with VIP, may directly participate in the regulation of immune function in the human.     

Effect of somatostatin-14 on duodenal mucosal bicarbonate secretion in guinea pigs

The role of somatostatin-14 in duodenal mucosal HCO ₃ ^– secretion was investigated in anesthetized, indomethacin-treated guinea pigs. Net HCO ₃ ^– output from the isolated, perfused (24 mM NaHCO₃ + 130 mM NaCl) proximal duodenum was measured during intravenous infusion (alone or in combination) of somatostatin-14, carbachol, vasoactive intestinal peptide (VIP), and prostaglandin E₂ (PGE₂). In homogenates of duodenal enterocytes, the effect of these agents on adenylate cyclase activity was studied. Basal duodenal HCO ₃ ^– secretion (3.5±0.2µmol/cm/10 min) was reduced dose dependently by somatostatin-14 (10^–11 mol/kg, 10^–9 mol/kg, and 10^–7 mol/kg). Carbachol, VIP, and PGE₂ (all 10^–8 mol/kg) increased basal duodenal HCO ₃ ^– secretion two- to threefold. Somatostatin-14 (10^–7 mol/kg) abolished the stimulatory effect of carbachol and VIP, but not that of PGE₂. Basal adenylate cyclase activity in isolated duodenal enterocytes (9.4±1.0 pmol cAMP/mg protein/min) was unaltered by somatostatin (10^–6 mol/liter) or carbachol (10^–3 mol/liter). VIP (10^–8 mol/liter) and PGE₂ (10^–7 mol/liter) increased adenylate cyclase activity two- to threefold, and these effects were unchanged by somatostatin-14 (10^–6 mol/liter). In conclusion, somatostatin-14 inhibits basal and carbachol- and VIP-stimulated duodenal HCO ₃ ^– secretion, and its mechanism of action is not via inhibition of adenylate cyclase activity in duodenal enterocytes.This study was supported by grants from the German-Israel Foundation for Scientific Research and Development (grant I-78-054.2/88), and the Israeli Ministry of Health.     

Interaction of vasoactive intestinal peptide with human blood mononuclear cells

The binding of vasoactive intestinal peptide (VIP) and the stimulation of adenylate cyclase were studied in mononuclear cells from human peripheral blood. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Binding studies suggested the presence of 2 classes of binding site: a class with high affinity (Kd = 2.4 X 10(-10)M) and low capacity (8 fmoles/10(6) cells), and a class with low affinity (Kd = 8.0 X 10(-8)M) and high capacity (800 fmoles/10(6) cells) at 15 degrees C. Secretin displaced [125I]VIP from the cells with a 400-fold lower affinity than VIP, but glucagon, somatostatin and insulin did not show any effect. VIP was a potent and efficient stimulator of cyclic AMP production. The stimulation was observed at a concentration as low as 3 X 10(-11)M and depended on time, temperature and pH. Maximal cyclic AMP production (4-fold above basal levels) was observed with 10(-9) M at 15 degrees. Half-maximal response was obtained at 10(-10)M VIP. Secretin was an agonist of VIP but exhibited a 7000 times lower potency. Peripheral blood mononuclear cells constitute an easily accessible and suitable system for the study of VIP action in different physiological and pathophysiological conditions.     

Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4⁺, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8⁺, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4⁺ or CD8⁺ cells or in the CD4⁺/CD8⁺ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4⁺ cells and an increase in CD8⁺ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4⁺, Leu 8^– (P<0.01) cells in all disease groups and an increase in CD4⁺, CD45RA⁺ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8⁺, Leu 8⁺ (P<0.01) cells were evident in the Crohn's disease group. Three-color FACS analysis revealed significant differences in the relative proportions of the defined T-cell subpopulations enumerated in the various groups as compared with the normal controls. These included a decrease in the proportions of Leu 8⁺, CD45RA⁺, (virgin) CD8⁺ T cells (P<0.05) and Leu 8^–, CD45RA⁺, (putative recall antigen helper) CD4⁺ T cells (P<0.01) in all patient groups as compared with normal controls. Conversely, an increase in the proportions of Leu 8^–, CD45RA⁺, (putative suppressor effector) CD8⁺ T cells (P<0.01), and Leu 8^–, CD45RA⁺, (function unknown) CD4⁺ T cells (P<0.05) was seen in all patient groups as compared with normal controls. CD patients but not UC or NIBD patients demonstrated a significant increase in the proportion of Leu 8^–, CD45RA⁺, (putative cytotoxic effector) CD8⁺ T cells (P<0.01). An increase in the ratio of Leu 8^–, CD45RA⁺, CD8⁺ (suppressor effector)/Leu 8⁺, CD45RA^–, CD8⁺ (putative cytotoxic effector) T cells was observed in all of the patient groups, but was most accentuated in those with Crohn's disease. Significant decreases in the ratios of Leu 8^–, CD45RA^–, CD4⁺ (putative nonspecific B cell helper)/Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper) T cells and Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper)/Leu 8^–, CD45RA⁺, CD4⁺ (function unknown) T cells were observed in all of the disease groups studied as compared with normal controls. These results suggest that the proportions of certain peripheral blood T-cell subpopulations are significantly altered in gastrointestinal inflammatory states. Further analysis of these T-lymphocyte subpopulations in the blood and tissues might provide valuable insights into immunological aberrations in inflammatory bowel diseases and might be of value in distinguishing among inflammatory diseases of the intestine.     

Interleukin-7 activates intestinal lymphocytes

Human intestinal lymphocytes, particularly intraepithelial lymphocytes, proliferate minimally to some agents, like mitogens and stimuli of the CD3 pathway. Thisin vitro finding may be due, in part, to a loss of factors foundin vivo. Three T-cell growth factors, IL-7, IL-9, and IL-12, were tested for their ability to stimulate the proliferation of intestinal lymphocytes. Both intraepithelial lymphocytes and lamina propria lymphocytes proliferated more vigorously to IL-7 than to IL-9 or IL-12, and only IL-7 increased stimulation through the CD3 pathway. The IL-7-induced response was IL-2-dependent: IL-2 receptors appeared on both intestinal lymphocyte types, and antibody to the IL-2 receptor blocked IL-7-induced proliferation. Both CD4⁺ and CD8⁺ T-cell subsets responded to this cytokine as shown by phenotype-depletion experiments and constancy in the CD4/CD8 ratios after culture with IL-7. In addition, the T-cell receptor and subsets responded equally well to IL-7. This newly described selective proliferative response of intestinal lymphocytes to IL-7, but not to IL-9 or IL-12, requires no preactivation and may enhance, growthin vivo.This work was supported by grants from the Crohn's & Colitis Foundation of America and the National Institutes of Health (DK42166).     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia

Summary The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16⁺, CD57⁺ and cytotoxic CD 8⁺) was studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57⁺, CD 16⁺ and cytotoxic CD 8⁺ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3⁺ lymphocytes, which were 1.7–27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4⁺ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57⁺ receptors in comparison with their peripheral blood CD 57⁺ lymphocytes or the CD57⁺ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.     

Exploration of the immunoreactivity of the Traditional Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide

AIM: To study the immunoreactivity of the Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide (VIP) and its therapeutic mechanism.METHODS: The immunoreactivity of the Chinese medicine Shenrouyangzhentang to VIP was detected in the plasma of 20 normal people and 20 patients with Piyinxu (Spleen Yin deficiency) using the radioimmunoassay (RIA) method.RESULTS: The maximum binding rate B₀/T was 53.29%, the non-specific binding rate N₀/T was 1.170%, and the VIP standard curve was Y = 0.81983 + 0.44319X - 0.28927X², R² = 0.990. The VIP content in Shenrouyangzhentang was 106.6 ng/L ± 20 ng/L), while it was 90.16 ng/L ± 15 ng/L in normal human plasma and 63.25 ng/L ± 11 ng/L in the plasma of Pixinxu patients. The difference between normal plasma and Pixinxu patient plasma was statistically significant (P < 0.05).CONCLUSION: The Chinese medicine Shenrouyangzhentang demonstrated VIP immunoreactivity similar to that of normal plasma. The (vasoactive intestinal polypeptide) VIP content in Pixinxu patient plasma was lower than that in healthy subjects (P < 0.05).     

VIP antagonists enhance excitatory cholinergic neurotransmission in the human airway

It has been reported that a low concentration of exogenously applied vasoactive intestinal peptide (VIP) suppresses the release of acetylcholine (ACh) from vagus nerve terminals in the ferret and feline trachea. There has been, however, no documentation of the prejunctional action of VIP in the human airway. We observed the effects of VIP and VIP antagonists on cholinergic excitatory neuro-effector transmission in the human bronchus to study the possible role of endogenous VIP on excitatory neurotransmission. In the human bronchus, VIP (10^–10 to 10^–7 M) showed no effect on either the contractions evoked by electrical field stimulation (EPS) or those evoked by ACh. To investigate the possible role of endogenous VIP on the human bronchus, we observed the effects of the VIP antagonists [4-Cl-D-Phe⁶,Leu¹⁷]-VIP and [Ac-Tyr¹,D-Phe²-]-GRF(1–29)-NH₂ on excitatory neuroeffector transmission. Both VIP antagonists (10^–8 M) significantly enhances the contractions evoked by EFS without affecting the ACh sensitivity of smooth muscle cells. These results indicate that VIP antagonists have a prejunctional action that enhances excitatory neurotransmission. This study suggests that endogenous VIP may suppresses ACh release from the vagus nerve terminals in the human airway. It is also suggested that exogenously applied VIP may be inactivated by some mechanism in the human airway. Offprint requests to: H. Aizawa.     

HCl-stimulated duodenal HCO 3 − secretion in conscious rat

Using an isolated loop of the proximal duodenum of conscious rats, the role of vasoactive intestinal peptide (VIP) in the duodenal HCO ₃ ^– response to HCl was examined, especially interactions with participating cholinoceptor mechanisms and prostaglandins. A 5-min perfusion with 150 mmol/liter HCl increased luminal VIP during 3 hr, with a peak output during and immediately after the acid challenge. The HCl-stimulated output was unaffected by atropine and hexamethonium, but was augmented by indomethacin from 13.6 (9.5–17.8) to 39 (20–85) fmol/cm/min. The HCO ₃ ^– secretion in response to graded doses of intravenous VIP (0.00625–6 nmol/kg/30 min) was dose-dependent to maximally 33.5±10.5 µmol/cm/hr. The HCO ₃ ^– secretion during a single intravenous infusion of VIP (12 nmol/kg/hr), 13.9±4.2 µmol/cm/hr, was unchanged by atropine, reduced to 10.0±3.5 µmol/cm/hr by hexamethonium, and augmented to 18.9±4.7 µmol/cm/hr by indomethacin. Exogenous VIP did not change the basal luminal output of PGE₂; neither did exogenous PGE₂ nor indomethacin affect the basal luminal output of VIP. HCl-induced increases in luminal outputs of VIP, substance P, and neurokinin A (the two latter with unknown roles) were differentially affected by atropine, hexamethonium, and indomethacin, indicating that the acid challenge released the peptides through controlled mechanisms. In conclusion, in the duodenal HCO ₃ ^– response to luminal HCl, VIP may have a stimulatory role, which partially depends on nicotinic, but not on muscarinic cholinoceptor mechanisms, and which is negatively modulated by prostaglandins.The study was supported by the Swedish Society of Medicine, The Professor Nanna Svartz Foundation, The Medical Research Council of the Swedish Life Insurance Companies, The Swedish Medical Research Council (7464), The Swedish Cancer Fund (2313) and by Funds of the Karolinska Institute.     

Vasoactive intestinal peptide

Dose—response characteristics of feline corpus circular muscle were studiedin vitro for three neuropeptides individually and with vasoactive intestinal peptide. Bombesin, substance P, and cholecystokinin-octapeptide each elicited concentration-dependent isometric contractions that were reduced by 10^–8 M or 10^–7 M vasoactive intestinal peptide (P<0.01). The concentration of each neuropeptide producing a half-maximal response was increased more than one logfold to 10^–6 M by vasoactive intestinal peptide. Tetrodotoxin blocked responses to bombesin (P<0.001) and reduced responses to substance P (P<0.05), but had no effect on responses to cholecystokinin-octapeptide (P>0.1). These results demonstrate inhibition of neuropeptide responses of gastric smooth muscle and support vasoactive intestinal peptide as an inhibitory regulator of gastric motor function.     

Distribution of vasoactive intestinal polypeptide and substance P receptors in human colon and small intestine

Vasoactive intestinal polypeptide (VIP) and substance P are found in neurons in the lamina propria and submucosa and muscularis propria of human small intestine and colon. VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle, and mononuclear cells. This study analyzes the distribution of[¹²⁵I]VIP binding and [¹²⁵]substance P in human colon and small intestine using autoradiographic techniques. [¹²⁵I]VIP binding was present in high density in the mucosal layer of colon and small intestine. [¹²⁵I]VIP binding was not significantly greater than nonspecific binding in smooth muscle layers or the lymphoid follicles. In contrast, [¹²⁵I]substance P binding was present in high density over the colonic muscle but was not present over the mucosal layer. In human colon cancer, [¹²⁵I]VIP grain density over the malignant tissue was only slightly higher than background. These autoradiographic studies of [¹²⁵I]VIP binding indicate that the highest density of VIP receptors was found in the small intestine and superficial colonic mucosa, whereas the density of substance P receptors was highest over the smooth muscle layers. These findings suggest a mismatch between immunochemical content of the peptide and autoradiographic density of the receptor.This work was supported by funds from the Research Service of the Veterans Administration.     

Decreased vasoactive intestinal peptide levels and glutathione depletion in acquired megacolon

We reported decreased vasoactive intestinal peptide levels in acquired megacolon. The origin of altered neuropeptide levels is unknown, but recent work suggested that tissue antioxidants may function as neuroprotectants. Our hypothesis was that altered levels of inhibitory neurotransmitters in human colon are associated with depletion of the tripeptide thiol, glutathione. Normal colon samples (N=10; from patients 41–80 years old) and acquired megacolon samples (N=10; from patients 31–98 years old) were obtained at surgery. Vasoactive intestinal peptide levels were decreased in muscularis externa from acquired megacolon (P=0.01), while there was a modest increase in NADPH diaphorase activity in muscularis externa from megacolon (P=0.10). Glutathione in acquired megacolon was detectable in muscularis externa from only five specimens (P<0.05), but was not significantly different (P>0.05) in the mucosal-submucosal layer. The results supported the presence of vasoactive intestinal peptide and NADPH diaphorase in distinct subpopulations of nerves in human colon. The results also supported the hypothesis that glutathione functions as a neuroprotectant in a subset of patients with acquired megacolon.Supported by VA Medical Research Funds.     

Gastrointestinal peptide hormones during postoperative ileus

The hypothesis was that postoperative ileus might be caused by a disturbed balance between the motor-stimulating hormones, motilin and substance P, and the motorinhibitory hormone, vasoactive intestinal polypeptide, and that octreotide might prevent this disturbance and so ameliorate the ileus. In 15 conscious dogs with chronic gastro-intestinal electrodes, electrical activity was recorded and blood was drawn for radioimmunoassay of motilin, substance P, and vasoactive intestinal peptide (VIP) during fasting and after a liquid meal. Ileus was then induced by celiotomy and intestinal abrasion. During and after operation, five dogs received 154 mM NaCl only, five dogs octreotide, 0.19 µg/kg/hr, and five octreotide, 0.83 µg/kg/hr. Plasma levels of motilin, substance P, and VIP were changed little by operation, but cyclical increases in plasma motilin, which occurred preoperatively during phase III of the interdigestive myoelectric complex, were completely abolished postoperatively during ileus, as was the complex itself. Octreotide ameliorated the ileus and restored the interdigestive complexes, but it decreased plasma motilin and did not restore the cyclic increases in motilin found in health, nor did it alter plasma substance P and VIP. In conclusion, octreotide ameliorates postoperative ileus, but it does not do so by increasing plasma motilin or substance P or decreasing plasma VIP.This work was supported by USPHS NIH grants DK18278 and DK07198, a grant from Sandoz Pharmaceuticals, and the Mayo Foundation.An abstract of this work has been published inGastroenterology 103:1382, 1992, and was presented at the biennial meeting of the American Motility Society, September 13–17, 1992, in Lake Tahoe, California.     

Catalytic Hydrolysis of VIP in Pregnant Women with Asthma

Rationale: The neuropeptide vasoactive intestinal peptide (VIP) is one of the physiologic mediators of non-adrenergic, non-cholinergic smooth muscle relaxation of the airway and an important modulator of innate and adaptive immune responses. VIP catalytic autoantibodies are increased in asthma and serum VIP level is decreased during acute exacerbation of asthma. The effect of pregnancy on asthma is variable and depends in part on the severity of pre-existing asthma, along with other physiological and pathophysiological changes. We hypothesized that hydrolysis of VIP by circulating catalytic VIP antibodies will be increased in pregnancy in patients with asthma. Study objective: To determine the level of catalytic autoantibodies to VIP in pregnant asthmatics compared to non-pregnant asthmatics and control pregnant women without asthma. Methods: We prospectively enrolled eight pregnant asthmatics (age, 26.5 ± 2.6 years; mean ± SEM), nine pregnant women without asthma (32.0 ± 3.0 years), seven non-pregnant women with asthma (25.0 ±1.9 years), and seven non-pregnant women without asthma (34.4 ± 2 years) into the study. VIP hydrolysis was performed in all subjects. Results: Immunoglobulin G (IgG) autoantibodies that catalyze the hydrolysis of vasoactive intestinal peptide (VIP) were present at greater levels in the blood of pregnant women with asthma (7.6 ± 1.1 pM VIP/6 h) compared to pregnant women without asthma (4.0 ± 0.5; p < 0.001), non-pregnant asthmatics (4.9 ± 0.9; p < 0.05) or non-pregnant women without asthma (1.9 ± 0.7; p < 0.05). Conclusion: An increase in the VIP hydrolyzing activity of IgG is independently associated with asthma and pregnancy. The autoantibodies hold the potential of affecting the pathophysiology of the airways in pregnant asthmatics.     

Vagal Control of the Release of Somatostatin,Vasoactive Intestinal Polypeptide,Gastrin-Releasing Peptide,and HC1 from Porcine Non-Antral Stomach

We studied the secretion of somatostatin and HO and the release of vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP) from isolated, vascularly perfused, porcine non-antral stomach. Electric vagus stimulation increased acid secretion and the release of VIP and GRP and inhibited somatostatin secretion as determined in the venous effluent. Atropine abolished the HC1 response and reversed the somatostatin inhibition to a three-fold increase, whereas GRP and VIP responses were unchanged. Both intra-arterial carbachol (10′⁶M) and GRP (10′⁸M) increased acid secretion and inhibited somatostatin secretion. VIP (10^_8M) increased somatostatin secretion and had no effect on acid secretion. By immunohistochemistry, somatostatin was localized to both open-type and closed-type cells equally spread in the various parts of the gastric glands without particular relation to the parietal cells. Numerous GRP- and VIP-immunoreactive nerve fibers were seen between the glands. It is concluded that the fundic and antral secretion of somatostatin, investigated in a previous study, are differently regulated. The relation of fundic somatostatin release to acid secretion seems to be complex.     

Augmented human-tumor-cytolytic activity of peripheral blood lymphocytes and cells from a mixed lymphocyte/tumor culture activated by interleukin-12 plus interleukin-2, and the phenotypic characterization of the cells in patients with advanced carcinoma

In this study, we evaluated the ability of combination regimens of interleukin-12 (IL-12) and interleukin-2 (IL-2) to induce effective killer cells against human tumors in vitro, in peripheral blood lymphocytes (PBL) from 15 cancer patients and mixed lymphocyte/tumor culture (MLTC) cells from 16 cancer patients, and carried out a phenotypic analysis of the cells responsible for the lysis of the human tumors. The freshly prepared PBL were cultivated with IL-2 alone or IL-12/IL-2 for 10 days [lymphokine-activated killer (LAK) cell generation system]. The MLTC cells (PBL cultured with mitomycin-C-treated allogeneic G-415 tumor cells for 3 days) were further cultivated with IL-2 or IL-12/IL-2 for 7 days [cytotoxic T lymphocytes (CTL) generation system]. The cytolytic activities of the lymphoid cells cultivated with IL-12/IL-2 were significantly augmented in both the LAK and CTL generation systems, as compared with those of cells treated with IL-2 alone. In the LAK generation system, the cytolytic activities of the cells cultivated with IL-12/IL-2 were significantly decreased by the method of negative selection of CD11b⁺ or CD56⁺ cells using immunomagnetic beads. The CD8⁺-depleted cells showed a slight decrease of activity. The killer cell activities of the CD4⁺-depleted cells remained unchanged. In the CTL generation system, the activity was markedly reduced by the elimination of the CD8⁺ or CD11b⁺ or CD56⁺ cells. The combined data suggested that IL-12/IL-2-induced killer effector cells in the LAK generation system were mainly of the natural killer (NK) type, comprising CD8^–CD11b⁺, CD8^– CD16b^–, CD3^–CD56⁺, and partly possible CD8⁺ CD11b^–T cells. CD8⁺ CD11b^–T cells mixed with cells of the NK type, comprising CD8^–CD11b⁺, CD8^– CD16b^– and CD3^–CD56⁺ cells, were the population of killer effector cells induced by IL-12/IL-2 in the CTL generation system.Abbreviations IL interleukin - LAK lymphokine-activated killer - CTL cytotoxic T lymphocytes - PBL peripheral blood lymphocytes - NK natural killer - MLTC mixed lymphocyte/tumor cell culture - TIL tumor-infiltrating lymphocytes     

The influence of VIP and epoprostenol on platelet CD62P expression and primary haemostasis in vitro

Human vasoactive intestinal peptide (VIP) and epoprostenol (prostacyclin) have vasodilatative effects in the pulmonary circulation. Both VIP and epoprostenol are successfully used to treat pulmonary hypertension in humans and experimental animal models. The positive effects of epoprostenol on the course of this disease are achieved through vasodilatation and inhibitory effects on platelet activity. Since VIP also binds specifically to platelets, we compared the in vitro effects of VIP and epoprostenol on platelet P-Selectin (CD62P) expression and primary haemostasis. Anti-aggregative effects of VIP (10^?6?mol and 10^?8?mol) and epoprostenol (50, 5 and 0.5?ng/ml) on platelets were determined by agonist-induced CD62P expression and in vitro bleeding time (PFA-100? system). Blood from healthy individuals was either incubated with epoprostenol, VIP or saline control and was analysed by whole blood flow cytometry and the PFA-100?. Prior to flow cytometric analysis, the platelets were stimulated with either arachidonic acid (AA) or adenosine diphosphate (ADP). Whole blood flow cytometry analysis showed that epoprostenol inhibited dose-dependently agonist-induced CD62P expression, whereas VIP did not inhibit CD62P expression. PFA analysis revealed substantial closure time prolongation by epoprostenol and again no effects of VIP. These results indicate that VIP, in contrast to epoprostenol, has no effect on platelet CD62P expression and primary haemostasis.     

Binding sites for peptide-histidine-isoleucine (PHI) on rat insulinoma-derived RINm5F cells

Specific binding sites for ¹²⁵I-labelled rat peptide-histidine-isoleucine (PHI) were identified on rat insulinoma-derived RINm5F cells. The concentrations of peptides producing half-maximal displacement of label were rat PHI, 0.36 ± 0.14 nM, vasoactive intestinal polypeptide (VIP), 0.38 ± 0.13 nM and secretin, approximately 0.2 μM. Glucagon and glucagon-like peptide-1(7–36)amide were without effect on binding. PHI and VIP produced dose-dependent increases in cAMP production in the cells that were significantly (P < 0.05) above unstimulated rates for ligand concentrations between 10⁻⁸ and 10 ⁻⁶ M. Both PHI and VIP produced a small but significant (P < 0.05) enhancement in the rate of release of immunoreactive insulin from the cells but the effect was not dose dependent.     

Inhibitory neuropeptides and intrinsic inhibitory innervation of descending human colon

The effects of aging on inhibitory neuropeptide concentrations and intrinsic inhibitory innervation of circular muscle were investigated using normal descending colon obtained at surgery. Immunoreactive vasoactive intestinal peptide, peptide histidine-methionine, met⁵-enkephalin, neuropeptide Y, and somatostatin were extracted from specimens of muscularis externa (patient ages: 19–84 years) and measured by radioimmunoassay. Intracellular electrical activity was recorded from strips of circular muscle (patients ages: 49–84 years) using glass microelectrodes; inhibitory junction potentials were evoked by electrical field stimulation. There were no significant differences (t tests:P>0.05) between neuropeptide concentrations in patients<70 years old (N=28) compared to patients70 years old (N=12). However, the amplitude of inhibitory junction potentials declined with increasing patient age (r=–0.58,P=0.02,N=16), with no change in resting membrane potentials (r=0.22;P>0.05). The decline in amplitude in women (r=–0.68,P=0.03,N=9) preceded the decline in men (r=–0.62,P=0.10,N=7). Age-related decline in inhibitory junction potentials could be related to decreased: density of inhibitory nerves, release of inhibitory neurotransmitter, density of binding sites for inhibitory neurotransmitter on smooth muscle, or a combination thereof. Alternatively, this decline might represent a change in interaction of inhibitory neurotransmitter with the smooth muscle membrane, such as a change in coupling of binding site with the potassium channel, decreased number of potassium channels, or altered permeability of the potassium channel.This study was supported in part by the National Institutes of Health (DK 17238 and DK 34988), and by VA Medical Research funds.