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L Eckhardt

Publications and source records attributed to L Eckhardt.

29 records · Page 2Linked to original sources
PubMed1991

Effects of cholecystokinin, cholecystokinin JMV-180 and GTP analogs on enzyme secretion from permeabilized acini and chloride conductance in isolated zymogen granules of the rat pancreas.

Previous studies have shown that hormonal activation of the Cl- conductance in pancreatic zymogen granules (ZG) is closely related to enzyme secretion from acinar cells. We have now examined the role of guanine nucleotides in stimulated and unstimulated protein secretion from isolated digitonin-permeabilized pancreatic acini and in the Cl- conductance of isolated ZG. Protein secretion from permeabilized isolated acini, measured at 0.1 mM Ca2+, increased with increasing cholecystokinin octapeptide (CCK-8) concentrations and decreased at high CCK-8 concentrations. The maximum secretion, approximately twice the control level, was reached at 1 nM CCK-8. The CCK analog, CCK JMV-180, which supposedly acts as an agonist on high-affinity CCK receptors and as an antagonist on low-affinity CCK receptors, stimulated maximum enzyme secretion at a CCK JMV-180 concentration of 0.1 microM and no decrease in secretion was observed at higher CCK JMV-180 concentrations, 0.1 mM guanosine 5'-[gamma-thio]triphosphate (GTP [S]) also increased the protein release by approximately twice that of the control and shifted the CCK-8 concentration causing maximum stimulation from 1 nM to 0.01 nM. GTP[S] concentrations greater than 0.1 mM inhibited protein release evoked by an optimal concentration of 1 nM CCK-8, 0.1 mM GTP[S] had no pronounced effect on the protein secretion stimulated by low concentrations of CCK JMV-180, but inhibited protein secretion evoked by CCK JMV-180 concentrations greater than 0.1 microM. This indicates that guanosine-nucleotide-binding proteins [G protein(s)] coupling to CCK receptors also mediate both CCK-induced increases and CCK-induced decreases of enzyme secretion at low and high CCK concentrations, respectively. ZG were prepared on a Percoll gradient from CCK-8-stimulated or CCK-JMV-180-stimulated and unstimulated acini. Their Cl- conductances were estimated in the absence of Ca2+ and in the presence of 1 mM EGTA from the rate of decrease in absorbance following addition of the K+ ionophore valinomycin as a measure of ZG osmotic lysis. The Cl- conductance in ZG from CCK-8-stimulated and CCK-JMV-180-stimulated acini was maximally activated at 1 pM and 10 nM respectively. At higher agonist concentrations, Cl- conductance was decreased. Direct addition of 10 microM GTP[S] to isolated ZG from unstimulated acini increased the rate of lysis by approximately 40% of the control value. This effect was approximately additive to that of CCK-8 or of CCK JMV-180 prestimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Ionic and osmotic dependence of secretion from permeabilised acini of the rat pancreas.

Many types of secretory granule have been observed to swell as a result of cell stimulation implying a degree of osmotic control, although the regulation of granule fusion with the apical plasma membrane is not clearly understood. In the present study we have investigated the ionic and osmotic dependency of basal and stimulated 3H-protein release from rat pancreatic acini, permeabilised by either digitonin or high voltage electric discharge. Acini were stimulated with either cholecystokinin-pancreozymin octapeptide (CCK-8), carbachol (CCh), or with phorbol ester (TPA) plus cAMP. Stimulated secretion was significantly reduced when 130 mmol/l Cl- in the buffer was replaced by I-, NO3-, SCN- or cyclamate-. Secretion in Cl- buffers was inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), by 40% of the control response. Neither Na+ nor N-methyl-D-glucamine+ could replace K+ in the buffer. Ba2+ and quinine, which block K+ conductance pathways, inhibited stimulated secretion by 50%. Finally, stimulated secretion from leaky cells was nearly abolished by doubling buffer osmolarity. The data suggest that when the cell is stimulated, a Cl- and a K+ permeability appear in the zymogen granule membrane and the ions enter down their electrochemical gradients. The increased intragranular osmolarity results in granular swelling which is intimately associated with secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Acetylcholine and cholecystokinin receptors functionally couple by different G-proteins to phospholipase C in pancreatic acinar cells.

We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTP gamma S but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP-analogue, 8-bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK- and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

The role of phosphatidylinositides in stimulus-secretion coupling in the exocrine pancreas.

Cell activation of different cell types is accompanied by receptor-mediated stimulation of phospholipase C and a consequent breakdown of phosphatidylinositol 4,5-bisphosphate. Evidence suggests that GTP-binding proteins are involved in this signal transduction mechanism, which couples receptors to phospholipase C. Both the hydrolysis products diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3) are intracellular messengers for cellular responses such as secretion, as illustrated by the pancreatic acinar cell. IP3 releases Ca2+ from a nonmitochondrial Ca2+ pool likely to be the endoplasmic reticulum (ER). This Ca2+ release leads to a transient rise in the cytosolic free Ca2+ concentration from approximately 100 to approximately 800 nmol/liter, by which enzyme secretion is initiated. For sustained secretion, Ca2+ influx into the cell is necessary to keep the cytosolic free Ca2+ concentration at a slightly elevated level. Activation of protein kinase C by DG and Ca2+ seems to play a major role in the second, sustained phase of secretion. Ca2+ reuptake into the ER and Ca2+ extrusion from the cell are achieved by (Ca2+ + Mg2+)-ATPase in both the ER and the plasma membrane as well as by an Na+/Ca2+ exchange in the latter. In the final step of exocytosis, protein phosphorylation by Ca2+-, DG-, and cAMP-dependent protein kinases is probably involved.

Animals↗
PubMed1986

Ca2+-, phorbol ester-, and cAMP-stimulated enzyme secretion from permeabilized rat pancreatic acini.

Enzyme secretion from the exocrine pancreas is stimulated by receptor-activated breakdown of phosphatidylinositol 4,5-bisphosphate and consequent rise of both inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which leads to Ca2+ release and to activation of protein kinase C, respectively. Another way involves receptor-mediated stimulation of adenylate cyclase and consequent rise of cAMP and activation of protein kinase A. In the present work we have studied direct stimulation, inhibition, and mutual interaction of these pathways on enzyme secretion from isolated rat pancreatic acini that had been permeabilized by treatment with saponin or digitonin. The data were compared with those obtained in isolated intact acini. The data show that with increasing free Ca2+ concentrations greater than 10(-6) M protein release increases in "leaky" but not in "intact" cells and is maximal at approximately 10(-3) M, increasing about twofold compared with that in the absence of Ca2+. In the presence of the acetylcholine analogue carbachol, this effect of Ca2+ is enhanced by about threefold in leaky cells and is also present in intact cells to a similar extent. cAMP and its analogues, dibutyryl cAMP (dbcAMP) and 8-bromo-cAMP stimulate protein release by about twofold in the presence of Ca2+ in leaky cells. In intact acini cAMP has no effect, and cAMP analogues stimulate enzyme secretion by about twofold in some but not all experiments. Similarly, forskolin, an activator of adenylate cyclases and inhibitors of cyclic nucleotide-dependent phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX) and R0 201724, stimulate protein release in permeabilized acini. The Ca2+-binding protein calmodulin has no effect on enzyme secretion, whereas the calmodulin antagonist trifluoperazine dihydrochloride stimulates protein release in leaky but not in intact acini. The activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates protein release in a Ca2+-dependent manner and enhances cAMP-induced secretion. The effects of carbachol, TPA, cAMP, and a combination of both TPA and cAMP are inhibited by the polyamine spermine in permeabilized cells. Spermine has no effect on carbachol-induced enzyme secretion in intact cells. The data suggest that enzyme secretion from pancreatic acinar cells is mediated by cAMP protein kinase A and by Ca2+ phospholipid protein kinase C in a Ca2+-dependent way and that interaction occurs between both pathways.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells.

ATP-dependent 45Ca2+ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl2 and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na+,K+)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca2+ uptake was maximal with 10 and 2 mumol/liter free Ca2+, and half-maximal with 0.9 and 0.5 mumol/liter free Ca2+. It was maximal at 3 and 0.2 mmol/liter free Mg2+ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg2+ was replaced by Mn2+ or Zn2+ ATP-dependent Ca2+ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn2+ could replace Mg2+ for Ca2+ uptake by 20%. Other divalent cations such as Ba2+ and Sr2+ could not replace Mg2+ in Ca2+ uptake. Ca2+ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca2+ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca2+ uptake. The sequence was K+ greater than Rb+ greater than Na+ greater than Li+ greater than choline+ in plasma membranes and Rb+ greater than or equal to K+ greater than or equal to Na+ greater than Li+ greater than choline+ for rough endoplasmic reticulum. The anion sequence was Cl greater than or equal to Br greater than or equal to 1 greater than SCN greater than NO3 greater than isethionate greater than cyclamate greater than gluconate greater than SO2(4) greater than or equal to glutarate and Cl- greater than Br greater than gluconate greater than SO2(4) greater than NO3 greater than 1 greater than cyclamate greater than or equal to SCN, respectively. Ca2+ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K+ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca2+ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca2+ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca2+ from the cell that might be involved in the regulation of the cytosolic Ca2+ level.

Adenosine Triphosphate↗
PubMed1984

Characterization of calcium uptake into rough endoplasmic reticulum of rat pancreas.

ATP-dependent Ca2+ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca2+ -specific electrode and 45Ca2+. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na+,K+(Mg2+)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared, 45Ca2+ uptake correlated with the RNA content and not with the Na+,K+(Mg2+)ATPase activity. The characteristics of nonmitochondrial Ca2+ uptake into leaky isolated cells and 45Ca2+ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg2+, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca2+ concentration of 2 and 2 mumol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration. 45Ca2+ uptake was dependent on monovalent cations (Rb+ greater than K+ greater than Na+ greater than Li+ greater than choline+) and different anions (Cl- greater than Br- greater than SO4(2-) greater than NO3- greater than I- greater than cyclamate- greater than SCN-) in both preparations. Twenty mmol/liter oxalate enhanced 45Ca2+ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca2+ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca2+ concentration to 0.4 mumol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca2+ concentration in pancreatic acinar cells.

Adenosine Triphosphate↗
PubMedDate not supplied

Reducing ultraviolet radiation exposure in children.

BACKGROUND: Project SUNWISE evaluated the effectiveness of a multicomponent intervention in reducing children's ultraviolet radiation (UVR) exposure. METHODS: Across four YMCAs, 48 aquatics classes (N = 169 children, mean age = 7) were randomly assigned to either the intervention or the control condition. The 6-week intervention included a UVR reduction curriculum presented at poolside by aquatics instructors and home-based activities for children and their parents. Outcome measures were (a) tanness-associated skin color dimensions assessed with a colorimeter, (b) specific daily solar protection behaviors of children as reported by parents, and (c) general solar protection behaviors. RESULTS: Controlling for intraclass clustering in all analyses, at posttest, no statistically significant between-group differences were found in tanness, daily solar protection scores, or general sunscreen use. The intervention group showed significantly greater general hat use relative to controls. CONCLUSIONS: The intervention failed to impact most of the outcome measures. Supplementing the behavior-focused intervention package with environmental supports may be warranted.

California↗
PubMedDate not supplied

Related effectiveness of continued, lapsed, and delayed smoking prevention intervention in senior high school students.

The relative effectiveness of continued, lapsed, and delayed smoking prevention intervention was tested with senior high school students. The original intervention was conducted during Grades 7 through 9, with significantly fewer intervention students reporting smoking than control students. The intervention was reintroduced in the 11th grade to one-half of intervention students (continued intervention), was withdrawn from the other half (lapsed intervention), and was initiated with one-half control students (delayed intervention). The 11th-grade smoking rates of these groups were compared to those of a fourth group, a continued control group. Results showed that continued control students reported significantly less smoking than continued control students and lapsed intervention students. Additionally, the delayed intervention group exhibited smoking rates lower than the lapsed intervention and continued control groups. This finding underscores the importance of continuing smoking prevention activities, as well as initiating these activities, in senior high school years.

Adolescent↗