Histamine-stimulated Cells Research Articles

Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte Differentiation.

Simple SummaryKonjac ceramide (kCer) is a unique molecular species of plant-type ceramide, and is a potential Sema3A-like ligand of Nrp1. kCer suppresses histamine-stimulated cell migration of HaCaT keratinocytes. This effect of kCer is not due to histamine-activated GPCRs, but rather to Sema3A-Nrp1 receptor binding. The present study focused on the ability of kCer to induce cell differentiation, in addition to its anti-migratory effects. We demonstrated that the effects of kCer on cell migration and cell differentiation are perpetuated by a cascade of crosstalk between pathways downstream of Nrp1 and GPCR in HaCaT cells.Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.

Pharmacological and Genetic Inhibition of Autophagy Decreases the Secretion of High Molecular Weight Von Willebrand Factor from Endothelial Cells in Vitro and in Vivo

The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Vasodilatory mechanism of levobunolol on vascular smooth muscle cells

Topical application of levobunolol hydrochloride, a β-adrenergic antagonist used for treatment of glaucoma, is reported to increase ocular blood flow. We studied the mechanism of levobunolol-induced vasodilation in arterial smooth muscle. The effects of levobunolol or other agents on isolated, pre-contracted rabbit ciliary artery were investigated using an isometric tension recording method. The effects of the same agents on intracellular free calcium ([Ca 2+] i) in cultured human aortic smooth muscle cells were also studied by fluorophotometry. Levobunolol relaxed ciliary artery rings that were pre-contracted with either high-K solution, 1 μM histamine, 10 μM phenylephrine, or 100 nM endothelin-1. The relaxation induced by levobunolol was concentration-dependent over the range of 10 μM to 0.3 mM. Inhibition of endothelial nitric oxide synthase or denudation of the endothelium did not affect this relaxation. Histamine-induced contractions were inhibited by the histamine H 1 antagonist pyrilamine. Radioligand binding experiments showed that levobunolol did not bind to the H 1 receptor. Further, histamine induced transient contraction in Ca 2+-free solution, and levobunolol inhibited this contraction by 74.6 ± 11.0%. In cultured smooth muscle cells in the presence of extracellular Ca 2+, levobunolol significantly inhibited the histamine-induced elevation of [Ca 2+] i. However, it did not inhibit the increase of [Ca 2+] i in histamine-stimulated cells incubated in Ca 2+-free solution. These results indicate that levobunolol may relax rabbit ciliary artery by two different mechanisms. First, the relaxation could be due to the blockade of Ca 2+ entry through non-voltage-dependent Ca 2+ channels. Second, levobunolol may change the Ca 2+ sensitivity of vascular smooth muscle cells.

The effect of n-3 PUFA on eNOS activity and expression in Ea hy 926 cells

The aim of this study was to evaluate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on NO synthase (eNOS) activation in Ea hy 926 endothelial cells. EPA or DHA (0–80 μM), added to the culture medium during 24 h, were dose-dependently incorporated into the cells. In control medium, eNOS activity (evaluated by the citrulline assay) and eNOS phosphorylation on Ser 1177 were correlated. They were increased by 10 μM histamine and prevented by 20 μM lysophosphatidylcholine (LPC). By contrast, EPA or DHA increased basal phosphorylation without affecting eNOS activity in non-stimulated cells, but dose-dependently decreased this activity in histamine-stimulated cells without modifying the phosphorylation level. Furthermore, EPA and DHA did not prevent the deleterious effects of LPC on histamine stimulation. In conclusion, incorporation of EPA and DHA could be deleterious for endothelial cells by deregulating the activation of eNOS and preventing NO liberation.

Comparative study of topical anti-allergic eye drops on human conjunctiva-derived cells: responses to histamine and IFNγ and toxicological profiles

The purpose of the study was to compare toxic effects and responses to histamine and IFN gamma associated with the use of some widely used anti-allergic eye drops commercially available today. For dynamic studies, the Wong-Kilbourne cell line was stimulated for 24 h with histamine or IFN gamma in the presence or absence of anti-allergic eye drops. Supernatants of histamine-stimulated cells were evaluated for the production of IL-6 and IL-8 by ELISA, while the expression of ICAM-1 was evaluated by flow cytometry on IFN gamma-stimulated cells. Toxicological assays were performed using cold light cytofluorometry: viability and apoptosis as well as reactive oxygen species (ROS) and O2(.)- production were assessed using neutral red, Hoechst/propidium iodide, H(2)-DCFDA and hydroethidine tests, respectively. Antihistamines reduced IL-6 release and presented dose-dependent inhibitory effects on IL-8 production. None of the eye drops decreased the basal or IFN gamma-stimulated expression of ICAM-1. Conversely, eye drops preserved with benzalkonium chloride (BAC) induced even higher ICAM-1 expression levels on IFN gamma-stimulated cells than did IFN gamma alone, whereas unpreserved drugs had no effect. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress. The ability of topical ocular anti-H(1) drugs to significantly reduce the production of IL-6 and IL-8 argues that they may help treat the inflammatory processes occurring in allergic ocular surface disorders. Nevertheless, preserved ophthalmic formulations may enhance epithelial conjunctival expression of ICAM-1 in the presence of a low inflammatory stimulus, such as IFN gamma, and displayed toxic as well as pro-oxidative effects on these cells. Therefore, BAC used as preservative might in part interfere with the potential anti-inflammatory properties of the active compound by modulating the immuno-inflammatory response of epithelial conjunctival cells.

Nitric oxide mediates histamine induced down-regulation of H2 receptor mRNA and internalization of the receptor protein (R1).

During agonist-dependent long-term stimulation of cells, histamine receptor subtypes are frequently down-regulated. However, the mechanisms underlying the modulation of receptor expression during long-term histamine stimulation have yet to be resolved. Based on our recently reported results showing an H1-mediated down-regulation of histamine H2 receptor mRNA in endothelial cells, our aim was to characterize the mechanism controlling rapid and long-term histamine-mediated modulation of H2 receptor expression in more detail. We were able to show that the histamine-induced down-regulation of H2 receptor mRNA and cell surface expression lasting for 24 h was accompanied by augmentation of the receptor protein level in the cytoplasmatic fraction of endothelial cells for this time period. Furthermore, changes in receptor protein levels in whole-cell lysate were negligible, indicating that the rapid and prolonged modulation of cell surface H2 receptor levels by histamine was regulated solely via internalization. The role of nitric oxide (NO) as a key mediator in histamine-stimulated cell responses was underlined by subsequent studies showing the attenuation of histamine-induced H2 receptor mRNA down-regulation and protein trafficking following NO synthase isozyme inhibition.

Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells.

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (P f) from 2.02 ± 0.3 × 10−4 to 16.37 ± 0.5 × 10−4 cm/s at 20°C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 ± 0.3% under basal condition and decreased by 50% to 1.2 ± 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, P f decreased by ∼35% in 20-min histamine-stimulated cells. Both P f and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.

Comparison of antiproliferative effects of 1-histamine-2 receptor antagonists, cimetidine, ranitidine, and famotidine, in gastric cancer cells

In the immune system, histamine is known to suppress cytotoxic T-lymphocytes and mitogen induced lymphocyte thymidine uptake, down-regulate some cytokines, and activate suppressor T-lymphocytes, and in the gastrointestinal system, histamine was reported to have trophic effects on gastrointestinal epithelial cells. Enhanced rates of cell proliferation by histamine are implicated in the pathogenesis of carcinogenesis. This study was designed since there is a lack of comparative data about the cell proliferations of histamine-2 receptor antagonist (H2-RA), cimetidine, ranitidine, and famotidine, in gastric cancer. KATO-III and AGS cell lines were used in this experiment. The concentrations of the histamine and cimetidine were 10 −5, 10 −8M, respectively and those of ranitidine and famotidine were 10 −6-10 −9M, respectively. Cell proliferation after drug treatment was evaluated by direct cell counting, [ 3H]thymidine incorporation, and MTT assay. Activities of ornithine decarboxylase (ODC), a rate limiting enzyme in polyamine synthesis, were measured after each drug treatment. Protein kinase A, a cAMP-dependent protein kinase system, was assayed using [α- 32p]ATP. Histamine showed statistically significant cell proliferating effects in a dose-dependent manner ( P < 0.01), the maximal effect in 10 −5M concentration. ODC activities were increased in accordance with the increment of cell numbers after histamine treatment. Cimetidine reversed the histamine-stimulated cell proliferation significantly, the maximal effect in 10 −5M concentration ( P < 0.01). Although ranitidine showed the tendency to attenuate the cell proliferation dose-dependently, but without statistical significance, famotidine did not show such an effect at all. cAMP-dependent protein kinase activities were significantly increased following 10 −5M histamine treatment, also reversed significantly by cimetidine co-administration ( P < 0.01). Beneficial clinical outcomes could be anticipated from cimetidine treatment in patients with gastric cancer by anti-proliferating effects against gastric cancer cells. These effects of H2-RA are likely to be mediated by specific interactions at the H2-receptor.

Refilling state of internal Ca 2+ stores is not the only intracellular signal stimulating Ca 2+ influx in human endothelial cells

To further analyse the role of the refilling state of internal Ca 2+ pools in the stimulation of Ca 2+ influx in human endothelial cells, we investigated the combined effect of thapsigargin (TG) and histamine on cytosolic Ca 2+ concentration ([Ca 2+] i) and inositol polyphosphate production. At normal extracellular Ca 2+ levels, TG induced a progressive and sustained elevation in [Ca 2+] i which was dose-dependently prevented by pretreatment with 1–10 μM histamine. Similarly, pretreatment with 0.1 and 1 μM TG suppressed histamine-induced Ca 2+ transients partially and totally, respectively. TG pretreatment did not alter the inositol triphosphate (IP 3) level liberated by histamine, but modified IP 3 metabolism by decreasing inositol biphosphate (IP 2) and increasing inositol monophosphate (IP i) contents. In the absence of Ca 2+ influx, 1 μM TG only induced a small transient increase in [Ca 2+] i whereas the Ca 2+ mobilization evoked by 10 μM histamine was unchanged. In both cases, the absence of any additional effect of either TG, histamine or 2 μM ionomycin indicated the complete depletion of Ca 2+ stores. The re-establishment of the transmembrane Ca 2+ gradient induced a transient rise in [Ca 2+] i. Its amplitude differed between histamine- and TG-treated cells. It was imposed by cell pretreatment and was selectively affected by changes in the membrane potential. At 5 mM external K +, the transient rise in [Ca 2+] i was more marked in histamine- than in TG-stimulated cells; this difference was suppressed by TG pretreatment. The presence of 130 mM external K + increased Ca 2+ entry in TG-treated cells but reduced it in histamine-stimulated cells. These results indicate that the refilling state of internal Ca 2+ stores does not constitute the single regulator of Ca 2+ influx. TG and histamine seem to activate Ca 2+ influx through distinct but interdependent pathways regulated by membrane potential.

Binding of epidermal growth factor to receptors in preparations of enriched porcine parietal cells and inhibition of aminopyrine uptake.

Preparations of isolated porcine gastric cells, enriched in parietal cells, were used to study binding of epidermal growth factor (EGF) to receptors and subsequent inhibition of [14C]aminopyrine uptake. EGF in concentrations from 10(-10) to 10(-7) M inhibited aminopyrine uptake stimulated by 10(-5) M histamine with an IC50 of 3 x 10(-10) M. [125]EGF bound in a saturable and specific manner to sites on cells in preparations containing 40-90% parietal cells. Mean apparent dissociation constant for the sites was 1.6 x 10(-9) M, with an average number of approximately 20,000 sites per cell. Endocytosis of ligand by parietal cells was limited, amounting to 10-20% of bound EGF after 1 h of incubation at 37 degrees C. Occupation of a fraction of the receptors caused a maximal reduction by 40% of aminopyrine uptake in histamine-stimulated cells, suggesting the occurrence of spare receptors. The results indicate the existence of specific receptors for EGF on porcine parietal cells exerting a regulatory influence on acid secretion.

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.

Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, [14C]aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor "physiological" responses at the same time within the same cell.

Gastric H + secretion: histamine (cAMP-mediated) activation of protein phosphorylation

Activation of H+ secretion by the gastric parietal cell involves major changes in morphology, metabolic activity and ion pathways of the secretory membrane. These changes are elicited by histamine binding to the H2 receptor, raising cAMP levels and presumably activating cAMP-dependent protein kinase. Concomitantly, the intracellular free Ca2+ concentration, [Ca2+]i, increases. Studies were performed to determine whether cAMP-mediated protein phosphorylation accompanies histamine activation of H+ secretion and to catalogue the major protein species serving as substrates for cAMP-dependent protein kinase in the parietal cell. 80% pure rabbit parietal cells, prepared by Nycodenz bouyant density centrifugation, were used. To investigate only cAMP-mediated effects, histamine-dependent changes in [Ca2+]i in these cells were abolished by depleting intracellular Ca2+ stores and performing experiments under Ca2+-free conditions. Acid secretion and steady-state levels of protein phosphorylation were then measured in unstimulated (cimetidine-treated) and histamine-stimulated cells. In intact parietal cells, concommitant with histamine stimulation of H+ secretion, increases in the level of protein phosphorylation were observed. Significantly changing phosphoproteins found in supernatant fractions showed apparent subunit sizes of approx. 148, 130, 47 and 43 kDa, and in microsomal fractions included those at approx. 130, 51 and 47 kDa. In parietal cell homogenates, using [gamma-32P]ATP, cAMP elicited significant phosphorylation of eight supernatant proteins and twelve microsomal proteins, which included the histamine-dependent phosphoproteins found in the intact parietal cell, except for the 51 kDa microsomal protein. As a working hypothesis, these proteins are involved in stimulus-secretion coupling in the parietal cell.

Gastric H + secretion: histamine (cAMP-mediated) activation of protein phosphorylation

Activation of H+ secretion by the gastric parietal cell involves major changes in morphology, metabolic activity and ion pathways of the secretory membrane. These changes are elicited by histamine binding to the H2 receptor, raising cAMP levels and presumably activating cAMP-dependent protein kinase. Concomitantly, the intracellular free Ca2+ concentration, [Ca2+]i, increases. Studies were performed to determine whether cAMP-mediated protein phosphorylation accompanies histamine activation of H+ secretion and to catalogue the major protein species serving as substrates for cAMP—dependent protein kinase in the parietal cell. 80% pure rabbit parietal cells, prepared by Nycodenz bouyant density centrifugation, were used. To investigate only cAMP-mediated effects, histamine-dependent changes in [Ca2+]i in these cells were abolished by depleting intracellular Ca2+ stores and performing experiments under Ca2+-free conditions. Acid secretion and steady-state levels of protein phosphorylation were then measured in unstimulated (cimetidine-treated) and histamine-stimulated cells. In intact parietal cells, concommitant with histamine stimulation of H+ secretion, increases in the level of protein phosphorylation were observed. Significantly changing phosphoproteins found in supernatant fractions showed apparent subunit sizes of approx. 148, 130, 47 and 43 kDa, and in microsomal fractions included those at approx. 130, 51 and 47 kDa. In parietal cell homogenates, using [γ-32P]ATP, cAMP elicited significant phosphorylation of eight supernatant proteins and twelve microsomal proteins, which included the histamine-dependent phosphoproteins found in the intact parietal cell, except for the 51 kDa microsomal protein. As a working hypothesis, these proteins are involved in stimulus-secretion coupling in the parietal cell.

Role of phosphoinositides in the regulation of endothelial prostacyclin production.

To elucidate the role of the phosphoinositide signal transduction system in endothelial endothelial prostacyclin production, endothelial cells from human umbilical veins previously labelled with 3H-inositol were incubated with thrombin or histamine. Water-soluble inositol phosphates were separated on anion exchange columns. Both agonists evoked transient bursts of inositol phosphate production with inositol trisphosphate peaking at 15 seconds in histamine-stimulated cells and at 60 seconds in thrombin-stimulated cells. The inositol phosphate production was closely linked to prostacyclin production. After stimulation, there was concurrent desensitization to prostacyclin production and formation of inositol phosphates. Arachidonic acid and the Ca2+-ionophore A23187 did not affect inositol phosphate production in concentrations sufficient to increase prostacyclin production 20-fold, and they did not affect desensitization to a subsequent thrombin stimulation. The phorbol ester 12-o-tetradecanoyl phorbol 13-acetate, a stimulator of protein kinase C, inhibited thrombin-induced generation of inositol phosphates, enhanced A23187-mediated prostacyclin production, and had complex effects on thrombin-mediated prostacyclin production, but had no effect on its production from extrinsic arachidonic acid. The current data suggest that production of inositol phosphates is a link in receptor stimulation of endothelial cells to produce prostacyclin and that associated activation of protein kinase C affects both the generation of second messengers and the release of arachidonic acid.

Stereological Analysis of the Parietal Cell During Acid Secretion and Inhibition

A stereological membrane analysis was carried out on parietal cells taken from canine fundic gastric mucosa during inhibition of histamine-activated acid secretion by cimetidine (3 mg per kg per hr), isoproterenol (1 μg per kg per min), and thiocyanate (0.2 mEq per min). Surface density (Sv) of secretory, tubulovesicular, and nutrient membranes was quantitated by stereological methods. of 118 parietal cells taken from resting mucosa, 5.7% appeared secreting and 13.6% of 165 histamine-stimulated cells retained their resting configuration. With histamine (1 μg per kg per min), secretory membrane increased and tubulovesicular membrane was reduced. Except for thiocyanate-inhibited cells, a linear relationship existed between secretory membrane Sv and acid output. Complete inhibition of histamine-stimulated acid secretion was produced by: cimetidine in 30 to 45 min, isoproterenol in 1.5 to 4 hr, and thiocyanate in 1.25 hr. Based on their ultrastructural response, a total of 88.6% of cimetidine-inhibited cells returned to resting in comparison to 51.1% of isoproterenol-inhibited cells and 26.4% of thiocyanate-inhibited cells. Thus, after treatment by cimetidine, an H2 receptor antagonist, secreting cells returned most quickly and completely to their resting ultrastructural state. Isoproterenol, a β adrenergic agonist, was a slower-acting inhibitor with an ultrastructural effect intermediate between those of cimetidine and thiocyanate. Both cimetidine and isoproterenol decreased secretory membrane Sv during acid inhibition. In contrast, thiocyanate-inhibited parietal cells showed no statistical decrease in Sv, although lumenal acid secretion ceased.

Postosmication of submucosal gland cells in the fowl proventriculus.

The technique of postosmication has been applied to resting and histamine-stimulated cells in the submucosal glands of the fowl proventriculus. In resting cells, the smooth endoplasmic reticulum, the mitochondria, and occasional vacuole-containing bodies become impregnated, but the Golgi apparatus, or ‘Dalton complex’ remains unaffected. In stimulated cells, the degree of impregnation of the smooth endoplasmic reticulum and vacuole-containing bodies is increased, and more mitochondria become heavily impregnated, but the Golgi apparatus remains unaffected. The significance of these observations is discussed.