Effect Of Secretion Research Articles

Co-localisation of functional nicotinic and ionotropic nucleotide receptors in isolated cholinergic synaptic terminals

The combination of immunological and microfluorimetric techniques has permitted the identification and analysis of the Ca 2+ influx responses in single rat midbrain cholinergic terminals. These terminals represent 22% of the total synaptosomal population and about 63% of them responded to nucleotides by a Ca 2+ influx. The nucleotide response distribution in cholinergic synaptic terminals is as follows; 22.4% to diadenosine pentaphosphate (Ap 5A), 24.7% to adenosine 5′-triphosphate (ATP) and 16.3% to both agonists. The ATP and Ap 5A are able to induce acetylcholine release in a dose- and calcium-dependent way, being the EC 50 values 0.22±0.1 μM and 1.5±0.1 μM respectively. Specific inhibitors can block this secretory effect. The studies of Ca 2+ influx responses in isolated single synaptic terminals have also permitted to demonstrate the wide co-expression of functional nicotinic and nucleotidic receptors. The percentage values of the terminals responding to both ATP/nicotine and Ap 5A/nicotine were 18.4% and 19.1%, respectively, considering the total population. Immunological studies also confirmed the presence of P2X 3 subunits and α4 and α7 nicotinic receptor subunits in about 36%, 30% and 20%, respectively, of the cholinergic terminals.

Chlormadinone acetate versus micronized progesterone in the sequential combined hormone replacement therapy of the menopause

Objective: The efficacy and safety of chlormadinone acetate (CA) versus micronized progesterone (P) were assessed in non-hysterectomized postmenopausal women. Materials and methods: This was a multicenter, randomized, parallel group study with a 6-month double-blind period followed by a 12-month open period. Patients were randomized to receive every month during 18 months percutaneous 17β-estradiol ( E 2) 1.5 mg/day from Day 1 to 24 of treatment cycle, combined from Day 11 to 24 to either CA 10 mg/day ( n=167) or P 200 mg/day ( n=169). Endometrial biopsy (EB, main analysis criterion) was performed at baseline, and at Day 18–24 of the 6th and 18th cycles. Results: At Month 6, EB did not evidence any hyperplasia. EB were inadequate for assessment in 24.5% and 47.5% of patients in the CA and MP groups, respectively. CA was found to be as protective as P (96.3% and 92.0% of success). However, the hormonal status of the endometrium differed ( P<0.001): a secretory endometrium was found in 81.5% of the CA patients, compared to 50.7% in the P group. These transformations resulted in predictable, cyclic bleeding in 94.5% of the CA patients, compared to only 62.3% of the P patients ( P=0.0001). Unscheduled bleeding, spotting and/or metrorrhagia, were more frequent under P than under CA (17.9% and 13.7%, respectively). The beneficial effects on hot flushes were more important in the CA group than in the P ( P<0.001). At Month 18, the biopsy and clinical results were similar to those obtained at Month 6. The safety profile, particularly the lipid one, was similar in both groups, except for drowsiness and dizziness, which were significantly more frequent under P than under CA. Conclusion: The progestative effects of CA on the endometrium and on menopause-related symptoms were at least as good as those of P. Moreover, CA resulted more often than P in secretory effects, and in satisfying bleeding patterns.

Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(-) secretion in human intestinal epithelial cells.

The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl(-)-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2 and the Ca(2+)-ATPase inhibitor thapsigargin, but not by the L-type Ca(2+) channel blocker verapamil or DIDS, the nonselective inhibitor of Ca(2+)-dependent Cl(-) transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca(2+). RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl(-) channel-2 but not for the Ca(2+)-dependent Cl(-) channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl(-) secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca(2+)-dependent Cl(-) secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.

Both serotonin and a nitric-oxide donor cause chloride secretion in rat colonocytes by stimulating cGMP

Background. Previous studies have demonstrated that an antagonist of nitric oxide synthase inhibits neurally mediated chloride secretion in response to serotonin (5-HT). The purpose of this study was to demonstrate that chloride secretion in rat colonocytes that were caused by stimulation of neural 5-HT receptors is mediated by way of a nitrergic pathway that involves the activation of guanylate cyclase. Methods. The nitric oxide (NO) donor, diethylenetriamine/NO (DNO), was added to an enriched suspension of rat colonocytes that were preloaded with 36Cl−. In parallel experiments, DNO (1 μmol/L) was added to cells that were pretreated with the specific inhibitor of soluble guanylate cyclase, NS2028 (2 μmol/L). In additional studies, the neural 5-HT3 receptor agonist, 2-methyl-5-HT (10 μmol/L), was added to the serosal surface of muscle-stripped sheets of rat colonic mucosa that were mounted in Ussing chambers under voltage clamp conditions, both in the absence and presence of NS2028 (20 μm). Results. DNO induced 18.0% ± 8.0% greater 36Cl− efflux than controls (P <.05; n = 14 animals). This efflux was abolished by previous treatment with NS2028. In the chamber experiments, 2-methyl-5-HT induced electrogenic chloride secretion that was significantly inhibited by previous treatment with NS2028 (2.2 ± 0.5 μA/cm2 vs 13.1 ± 2.1 μA/cm2; P <.001; n = 9 animals). Conclusions. The predominant secretomotor neurotransmitter that mediates the chloride secretory effects of 5-HT in vitro is nitric oxide. Both the secretory effect initiated at the 5-HT3 receptor on enteric neurons and at the NO− receptor on the rat colonocytes are mediated through the activation of intracellular guanylate cyclase and the production of cyclic guanosine monophosphate. (Surgery 2001;130:236-41.)

Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbit-rabbit hybridoma.

The generation of stable rabbit-rabbit hybridomas is now possible by the recent development of a rabbit fusion partner. The ability to generate rabbit monoclonal antibodies (MAbs) can be advantageous because these rabbit immunoglobulins tend to exhibit higher affinity than murine MAbs. Furthermore, it has been observed that, in general, rabbits will elicit an immune response to antigens of limited immunogenicity in mice. Unfortunately, these rabbit-rabbit hybridomas secrete only 200 ng/mL to 5 microg/mL of immunoglobulin, which may limit larger scale production of rabbit antibodies. This study sought to determine if interleukin 6 (IL-6), which has been reported to have proliferative and secretory stimulating effects on some murine hybridomas, had any effect on a rabbit cell line that secretes a monoclonal IgG specific for estradiol. The results demonstrated that recombinant human IL-6 had a dose-dependent enhancing effect on the IgG secretion of the rabbit-rabbit hybridoma. The enhancing effect was consistent when the cells were continuously passed in the presence of IL-6. However, IL-6 did not affect the growth of the hybridoma. In contrast, no discernible effect was accomplished with recombinant mouse IL-6. Furthermore, no basal IL-6 activity was detected in the rabbit hybridoma extracellular medium. The IL-6 enhancement effect observed in this study may help to increase the immunoglobulin yield of rabbit hybridomas and to assist in the understanding of the mechanism(s) behind the lowered secretion level.

Prenatal and Postnatal Ovine Adrenal Cell responses to Prostaglandin E2

To examine the secretory effect of prostaglandin E(2) (PGE(2)) and ACTH on the adrenal glands of prenatal and postnatal sheep. Immunocytochemistry was used to examine the adrenal cortex and medulla for 17alpha-hydroxylase and tyrosine hydroxylase immunoreactivity. Microphysiometric technique was used to measure [H(+)] after exposure of whole dispersed prenatal and postnatal adrenal glands to PGE(2), ACTH, or both. Immunocytochemistry showed many cortical-type cells in all adrenal medullae and many medullary-type cells in fetal adrenal cortices. Maximum H(+) responsiveness to PGE(2) decreased with increasing age. The developmental age-related pattern of maximum percentage change in [H(+)] during ACTH exposure was similar to previous findings with cortisol production as the endpoint. ACTH stimulated H(+) production at 80 days' gestation and at all ages greater than 125 days' gestation (P <.05). The molar concentration of ligand required to elicit a response that was 50% of maximum response (EC(50)) for the ACTH response was lower in fetuses than in newborn lambs (<1 day and 3 days old), but there was no change in EC(50) for PGE(2) across the ages studied. Adrenal cell response to ACTH after prior ACTH and PGE(2) exposure was higher (P <.05) compared with ACTH after ACTH or ACTH alone at 110 days' gestation only and was lower in 3-day-old lambs. Based on the ACTH results, microphysiometry was a valid method for investigating dispersed adrenal cell physiology. Prostaglandin E(2) stimulated dispersed adrenal cells during the mid-gestation ACTH refractory period, but this effect decreased with increasing age. Prostaglandin E(2) sensitized adrenal cells to ACTH at 110 days' gestation but inhibited ACTH effects at postnatal day 3.

Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats.

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 microg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.

PVN lesions prevent the endothelin 1-induced increase in arterial pressure and vasopressin.

Endothelin (ET) acts within the central nervous system to increase arterial pressure and arginine vasopressin (AVP) secretion. This study assessed the role of the paraventricular nuclei (PVN) in these actions. Intracerebroventricular ET-1 (10 pmol) or the ET(A) antagonist BQ-123 (40 nmol) was administered in conscious intact or sinoaortic-denervated (SAD) Long-Evans rats with sham or bilateral electrolytic lesions of the magnocellular region of the PVN. Baseline values did not differ among groups, and artificial cerebrospinal fluid (CSF) induced no significant changes. In sham-lesioned rats, ET-1 increased mean arterial pressure (MAP) 15.9 +/- 1.3 mmHg in intact and 22.3 +/- 2.7 mmHg in SAD (P < 0.001 ET-1 vs. CSF) rats. PVN lesions abolished the rise in MAP: -0.1 +/- 2.8 mmHg in intact and 0.0 +/- 2.9 mmHg in SAD. AVP increased in only in the sham-lesioned SAD group 8.6 +/- 3.5 pg/ml (P < 0.001 ET-1 vs. CSF). BQ-123 blocked the responses. Thus the integrity of the PVN is required for intracerebroventricularly administered ET-1 to exert pressor and AVP secretory effects.

Involvement of gastrin in gastric secretory and protective actions of N-alpha-methyl histamine

Nα-methylhistamine (Nα-MH) is one of an unusual metabolite of histamine that was found in Helicobacter pylori-infected stomachs and is believed to interact with specific histamine H 1, H 2 and H 3-receptors to stimulate gastric acid secretion and gastrin release from isolated G-cells but the effects of Nα-MH on gastric mucosal integrity have been little studied. This study was designed; (1) to compare the effect of exogenous Nα-MH with that of standard histamine on gastric secretion and plasma gastrin levels in rats equipped with gastric fistula (series A); and (2) to assess the action of Nα-MH on gastric lesions induced by 100% ethanol (series B) in rats with or without removal of antral portion of the stomach (antrectomy). Rats of series B were pretreated intragastrically (i.g.) or intraperitoneally (i.p.) with Nα-MH or histamine (0.1–2 mg/kg) 30 min prior to 100% ethanol (1.5 ml, i.g.) with or without: (1) vehicle (saline); (2) RPR 102681 (30 mg/kg i.p.), to block CCK-B/gastrin receptors; and (3) ranitidine (40 mg/kg s.c.) to inhibit histamine H 2-receptors. The area of gastric lesions was determined planimetrically, gastric blood flow (GBF) was assessed by H 2-gas clearance method and venous blood was collected for determination of plasma gastrin levels by radioimmunoassay (RIA). Nα-MH and histamine dose-dependently increased gastric acid output (series A); the dose increasing this secretion by 50% (ED 50) being 2 and 5 mg/kg i.g or i.p., respectively, and this effect was accompanied by a significant rise in plasma gastrin levels. Both, Nα-MH and histamine attenuated dose-dependently the area of gastric lesions induced by 100% ethanol (series B) while producing significant rise in the GBF and plasma immunoreactive gastrin increments. These secretory, protective, hipergastrinemic and hyperemic effects of Nα-MH and histamine were completely abolished by antrectomy, whereas pretreatment with RPR 102681 attenuated significantly the Nα-MH and histamine-induced protection against ethanol damage and accompanying hyperemia. Ranitidine, that produced achlorhydria and a further increase in plasma gastrin levels, failed to influence the Nα-MH- and histamine-induced protection and accompanying rise in the GBF. We conclude that (1) Nα-MH stimulates gastric acid secretion and exhibit gastroprotective activity against acid-independent noxious agents in the manner similar to that afforded by histamine; and (2) this protection involves an enhancement in the gastric microcirculation and release of gastrin acting via specific CCK-B/gastrin receptors but unexpectively, appears to be unrelated to histamine H 2-receptors.

A reconsideration of the evidence for Escherichia coli STa (heat stable) enterotoxin-driven fluid secretion: a new view of STa action and a new paradigm for fluid absorption.

A review of the evidence for Escherichia coli STa causing fluid secretion in vito leads to the conclusion that the concept of STa acting through enhanced chloride secretion in order to derange intestinal function is unproven. However, a consistent effect of STa in the small intestine is on Na+/H+ exchange, leading to interruption of luminal acidification. A model for the action of STa, involving inhibition of Na+/H+ exchange, is proposed which explains the ability of STa to reduce absorption in vito but its inability to cause secretion in vito in contrast to its apparent secretory effect in vitro. The apparent ability to demonstrate secretion in vitro is shown to derive from methodologies which do not involve measurement of mass transport of water but instead, infer it from in vitro and in vivo proxy measurements. The in vitro demonstration of notional secretion after STa exposure can be reconciled with the proposed new model for fluid absorption in that cell swelling is argued to arise as a transient consequence of STa challenge followed by regulatory volume decrease. Evidence for this derangement model is presented in the form of observations derived from acute in vivo physiological studies and clinical studies on patients without the exchanger. This process of appraisal of the evidence for the mechanism of action of STa has led to a new model for fluid absorption. This is based on the formation of hypotonicity at the brush border luminal surface rather than hypertonicity within the lateral spaces as required by the present standing gradient model of fluid absorption. Evidence from the literature is presented for this new paradigm of water absorption, which may only be relevant for small intestine and other tissues that have Na+/H+ exchangers in contact with HCO-3-containing solutions but which may also be generalizable to all mammalian absorbing epithelial membranes.

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol.

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.

Involvement of Cellular Calcium in Exocrine Pancreatic Insufficiency during Streptozotocin-Induced Diabetes Mellitus

This study investigates the effects of the islet hormones insulin (Ins), glucagon (Glu), and somatostatin (Som) with nerve stimulation (EFS) acetylcholine (ACh) and cholecytokinin-octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i in the pancreas of age-matched control and diabetic rats. Either Ins, Glu or Som elicited small increases in amylase secretion from the pancreas of age-matched control animals compared to a much larger increase in amylase secretion with either EFS, ACh or CCK-8. Combining the islet hormones with either EFS, ACh or CCK-8 resulted in marked potentiation of amylase output. In the diabetic pancreas, the islet hormones had no effect on amylase secretion compared to diabetic control. Moreover, either EFS, ACh or CCK-8 evoked a much smaller increase in amylase output compared to age-matched control. In addition, the islet hormones failed to potentiate the secretory effects of either EFS, ACh or CCK-8. In fura-2 loaded acinar cells from age-matched control pancreas either Ins or Glu elicited a small increase in [Ca2+]i whereas Som had no effect. Both ACh and CCK-8 evoked large increases in [Ca2+]i compared to control. Combining either Ins, Glu or Som with either ACh or CCK-8 resulted in a marked elevation in [Ca2+]i compared to the responses obtained with either the islet hormones, ACh or CCK-8 alone. In diabetic fura-2 loaded pancreatic acinar cells, the islet hormones had no effect on [Ca2+]i compared to control and moreover, the responses were much smaller than those obtained in acinar cells from age-matched control. Both ACh and CCK-8 induced large increases in [Ca2+]i in diabetic acinar cells. However, combining the islet hormones with either ACh or CCK-8 failed to enhance [Ca2+]i compared to the reponses obtained in acinar cells from age-matched control. The results suggests that [Ca2+]i homeostasis is deranged during diabetes mellitus and this in turn is probably associated with reduced pancreatic amylase secretion.

δ-opioid receptors inhibit neurogenic intestinal secretion evoked by mast cell degranulation and type I hypersensitivity

Histamine and the mast cell degranulator, compound 48/80 produced elevations in short-circuit current, an electrical measure of active anion secretion, across porcine ileal mucosa sheets mounted in Ussing chambers. Luminally-applied β-lactoglobulin produced similar effects in mucosal sheets from cow’s milk-sensitized pigs. Their secretory effects were attenuated by blockers of H 1-histamine receptors, neuronal conduction or epithelial Na +/K +/Cl − cotransport. The δ-opioid agonist [ d-Pen 2, d-Pen 5]enkephalin suppressed mucosal responses to these substances in a naltrindole-reversible manner. Furthermore, submucosal mast cells and δ-opioid receptor-immunoreactive nerve fibers were observed in close juxtaposition. Intestinal neural pathways linking immediate hypersensitivity to secretory host defense appear to express inhibitory δ-opioid receptors.

D-mannoheptulose uptake and its metabolic and secretory effects in human pancreatic islets.

D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.

Mechanisms of Cholera Toxin‐Induced Diarrhea

In the pathogenesis of cholera, cyclic adenosine monophosphate, 5-hydroxytryptamine, prostaglandins, and the function of neuronal structures have been implicated. To elucidate the role of different isoforms of cyclooxygenase (COX)-1 and COX-2, selective COX-2 inhibitors were used. The selective COX-2 inhibitors NS-398 and DFU completely suppressed cholera toxin-induced prostaglandin E2 biosynthesis and caused a dose-dependent inhibition of cholera toxin-induced fluid secretion in the rat jejunum in vivo. Constitutive expression of COX-1 but also of COX-2 mRNA was found in mucosal scrapings of the rat jejunum. Cholera toxin had no effect on COX-1 as well as COX-2 mRNA expression. Treatment of rats with dexamethasone did not effect cholera toxin-induced prostaglandin E2 biosynthesis and did not influence the expression of COX-2 mRNA, further substantiating that cholera toxin does not cause an induction of COX-2 mRNA. Treatment of rats with E. coli lipopolysaccharide caused a marked increase in COX-2 mRNA expression that was inhibited by dexamethasone. In conclusion, the results provide evidence that cholera toxin, in addition to other mediators, uses prostaglandin E2 to exert its secretory effect and that in the case of cholera toxin prostaglandins are metabolized via COX-2.

Cholecystokinin does not affect the pancreatic contents of epidermal growth factor or its receptor.

Cholecystokinin (CCK) is a hormone with well-known secretory and trophic effects on the pancreas. This also is true for epidermal growth factor (EGF), which acts in a paracrine and autocrine way. The aim was to study the influence of CCK on cell proliferation in rat pancreas with special reference to the expression of EGF, the EGF receptor, and phosphorylated tyrosine. Twenty-four male Sprague-Dawley rats received either one single injection, or injections twice daily for 3 days of 6 microg sulfated CCK-8 (CCK-8S) subcutaneously in the neck. The same number of rats received injections of 1% bovine serum albumin (BSA) in the same way. The rats were killed 1, 3, or 6 hours after the last injection. One hour before killing, they received 50 mg/kg of bromodeoxyuridine (BrdU) intraperitoneally. Plasma was collected for analysis of CCK. The pancreas was dissected, and in situ hybridization using a probe for EGF mRNA was performed for semiquantification of gene expression. Immunocytochemistry using antibodies against the EGF receptor and phosphotyrosine was performed to examine the expression of the proteins, and against BrdU for measuring the cell proliferation. A single injection of CCK-8S led to hyperCCKemia at 1 and 3 hours afterward. After 6 hours, plasma CCK had returned to the same levels as in control rats. The cell proliferation was unaffected. The rats that received CCK-8S injections for 3 days still had hyperCCKemia 6 hours after the last injection. The cell proliferation was increased by CCK, as indicated by the BrdU labeling. However, neither body weight nor pancreatic weight was affected. In controls, EGF was expressed all over the gland, but its receptor and phosphotyrosine were expressed only in ductal cells and in the islet cells of endocrine pancreas. There was no difference in the pancreatic staining of EGF, its receptor, or phosphotyrosine at the different time points studied. There was no difference in the staining of EGF and its receptor between CCK-8S- and BSA-treated animals, but phosphotyrosine staining was detectable in acinar cells after 3 days of CCK-8S injections. Thus CCK-8S causes hyperCCKemia with ensuing enhanced cell proliferation in rat pancreas. This effect on the cell proliferation seems to be a direct effect of CCK and not mediated by changes in the tissue levels of EGF or its receptor.

Insulinotropic effect of Citrullus colocynthis fruit extracts.

Infusions of Citrullus colocynthis Schrad. (Cucurbitaceae) fruits are traditionally used as antidiabetic medication in Mediterranean countries, but to our knowledge no studies have been undertaken so far to determine the possible mechanisms involved in the antidiabetic properties of the fruit. The present study was designed to investigate whether these fruits possess insulinotropic effects. For this purpose, different extracts of Citrullus colocynthis seed components were obtained: RN II (crude extract), RN VI (hydro-alcoholic extract), RN X (purified extract) and RN XVII (beta-pyrazol-1-ylalanine), the major free amino acid present in the seeds. The insulin secretory effects of these different extracts were evaluated in vitro in the isolated rat pancreas and isolated rat islets in the presence of 8.3 mM glucose. All tested extracts, when perfused for 20 min at 0.1 mg/ml, immediately and significantly stimulated insulin secretion. This effect was transient. In addition, the purified extract (RN X) provoked a clear dose-dependent increase in insulin release from isolated islets. Moreover, a significant and persistant increase in pancreatic flow rate appeared during RN VI, RN X and RN XVII perfusions. In conclusion, our results show that different Citrullus colocynthis seed extracts have an insulinotropic effect which could at least partially account for the antidiabetic activities of these fruits.

Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation?

In addition to its contractile properties, airway smooth muscle may contribute to the pathogenesis of asthma by increased proliferation, and by the expression and secretion of pro-inflammatory cytokines and mediators. Studies of airway smooth muscle cells in culture have shown that many mitogenic mediators can induce proliferation, and that these may therefore, contribute to the increase in airway smooth muscle mass observed in asthma. Other mechanisms for airway smooth muscle proliferation include the interaction with inflammatory cells such as T-cells and eosinophils. Airway smooth muscle cells may also be a source of inflammatory mediators and cytokines, in particular chemokines, thus implicating airway smooth muscle cells as contributors to the inflammatory mechanisms of asthma. The pro-activating signals for converting airway smooth muscle cells into a proliferative and secretory cell in asthma are unknown, but may include viruses and immunoglobulin E. Airway smooth muscle contractility may also be altered in response to inflammation. Airway smooth muscle cells may play an important interactive role with inflammatory and other structural cells, contributing to inflammation, injury and repair of the airways. Such a recognition makes it imperative to consider the airway smooth muscle as a target of therapeutic drugs for suppressing not only the contractile but also the proliferative and secretory effects of asthma.

Residual Oil Fly Ash Induces Cytotoxicity and Mucin Secretion by Guinea Pig Tracheal Epithelial Cells via an Oxidant-Mediated Mechanism

Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 μg/cm2 ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

Metabolic and K(ATP) channel-independent actions of keto acid initiators of insulin secretion.

Insulin-releasing effects of 2-ketobutyric acid (KB), 2-ketoisocaproic acid (KIC), 2-keto-3-methylvaleric acid (KMV), and 3-phenylpyruvic acid (PP) were examined by using clonal beta cells. Whereas KIC, KMV, and PP dose-dependently initiated insulin secretion and potentiated the effects of 4.2-16.7 mM glucose, equimolar KB was without effect. Transport inhibition by using 10 mM valine, isoleucine, 2-cyano-3 hydroxycinnamate or 2-cyano-4 hydroxycinnamate, or metabolic inhibition by 15 mM mannoheptulose, 5 mM sodium azide, 5 mM sodium cyanide, or removal of HCO3 reduced the secretory effects of KIC, KMV, and PP. Whereas K+ depletion reduced keto acid-induced insulin output, depolarizing concentrations of L-leucine and L-arginine potentiated the keto acid-induced effects. Under depolarizing conditions (25 mM KCI and 16.7 mM glucose), 10 mM KIC, KMV, or PP induced insulin secretion, suggesting K(ATP) channel-independent actions. Furthermore, the K(ATP) channel opener diazoxide reduced, but did not abolish, the keto acid-induced effects. However, voltage-dependent Ca2+ channel blockade with verapamil or removal of extracellular Ca2+ abolished keto acid-induced insulin release. Collectively, these results indicate that KIC, KMV, and PP initiate insulin secretion at least partially independently of K(ATP) channel activity, through both mitochondrial metabolism and regulation of Ca2+ influx.