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J Gromada

Publications and source records attributed to J Gromada.

49 records · Page 3Linked to original sources
PubMed1996

Membrane potential and cytosolic free calcium levels modulate acetylcholine-induced inositol phosphate production in insulin-secreting BTC3 cells.

Effects of membrane potential and cytosolic free Ca2+ concentrations ([Ca2+]i) on acetycholine (ACh)-induced inositol phosphate production were investigated in insulin secreting betaTC3 cells. ACh (10 microM) caused a rapid inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and increase in [Ca2+]i reaching a maximum within 5 s. The rise in Ins(1,4,5)P3 production was reduced by 79 +/- 5% when [Ca2+]i was kept low in cells loaded with the Ca2+ chelator BAPTA. The ACh-evoked Ins(1,4,5)P3 production also depended on the membrane potential as it was reduced by 31 +/- 6% in cells hyperpolarized by diazoxide, an opener of ATP-sensitive K+ channels. The Ca2+ ionophore ionomycin caused a rapid increase in [Ca2+]i and in the cellular Ins(1,4,5)P3 content. We conclude that stimulation-induced changes in membrane potential and [Ca2+]i play an important role in controlling Ins(1,4,5)P3 production in insulin-secreting betaTC3 cells.

Acetylcholine↗
PubMed1996

Desensitization of glucagon-like peptide 1 receptors in insulin-secreting beta TC3 cells: role of PKA-independent mechanisms.

1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin-secreting beta TC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L-type Ca(2+)-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+]i evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+]i disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i. 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+]i is initiated by Ca(2+)-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP-1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca(2+)-dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP-1 receptors but that other (PKC-independent) mechanisms also participate in this process.

Animals↗
PubMed1995

Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-trisphosphate and stored calcium in rat parotid acini. ATP as a co-transmitter in the stimulus-secretion coupling.

The effect of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and inositol phosphate production following stimulation with the muscarinic cholinergic agonist acetylcholine (ACh) was investigated in isolated rat parotid acinar cells. Stimulation of rat parotid acinar cells with ATP4- results in a rise in [Ca2+]i that is due to influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Stimulation with purinergic agonists revealed that both influx as well as Ca2+ release from intracellular stores was mediated through activation of P2z receptors. The Ca2+ mobilization from intracellular stores was due to production of Ins(1,4,5)P3 and was inhibited by U73122, an inhibitor of phospholipase C-coupled processes. Under Ca(2+)-free conditions ATP4- caused a dose-dependent inhibition (IC50 = 8 microM) of the ACh-evoked Ca2+ release. The inhibitory effect of ATP4- is due to activation of the P2z purinoceptors, which results in a strong reduction in the ACh-induced inositol phosphate production. Prestimulation with 100 microM ATP4- reduced the amount of Ins(1,4,5)P3 formed after maximal ACh stimulation by 91%. In conclusion, the inhibitory effect of ATP4- on the ACh-mediated response is due to interactions of the activated P2z receptor with the phospholipase C-coupled processes underlying the muscarinic cholinergic response.

Acetylcholine↗
PubMed1995

Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release.

The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.

Acetylcholine↗
PubMed1995

Cyclic ADP-ribose and inositol 1,4,5-triphosphate mobilizes Ca2+ from distinct intracellular pools in permeabilized lacrimal acinar cells.

In permeabilized lacrimal acinar cells, cyclic ADP-ribose (cADP-ribose) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) release Ca2+ in a dose dependent manner from distinct thapsigargin-sensitive Ca2+ pools. Ryanodine specifically blocks the Ca2+ response to cADP-ribose, whereas heparin strongly reduces the response to Ins(1,4,5)P3 application. GTP causes a rapid Ca2+ release by a ryanodine- and heparin-insensitive mechanism and potentiates Ins(1,4,5)P3 but not cADP-ribose evoked Ca2+ release. It is estimated that cADP-ribose can release 16 mumol Ca2+/l cells, whereas Ins(1,4,5)P3 can mobilize 55 mumol Ca2+/l cells. The results suggest that cADP-ribose and Ins(1,4,5)P3 release Ca2+ from distinct internal stores and that a third Ca2+ pool exists which can selectively interact with the Ins(1,4,5)P3-sensitive Ca2+ store by a GTP-mediated process.

Adenosine Diphosphate Ribose↗
PubMed1995

The release of intracellular Ca2+ in lacrimal acinar cells by alpha-, beta-adrenergic and muscarinic cholinergic stimulation: the roles of inositol triphosphate and cyclic ADP-ribose.

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1-4 phosphate groups, resulting in release of Ca2+ from intracellular stores. Stimulation with the alpha-adrenergic agonist phenylephrine, however, causes a release of Ca2+ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca2+ rise was completely inhibited by 100 microM ryanodine. Adrenaline (causing activation of both alpha- and beta-adrenergic receptors) induces a Ca2+ release with inositol phosphate synthesis identical to that occurring in the beta-adrenergic response. Thus, the signalling pathway for alpha-adrenergic stimulation occurs via a path different from that which releases Ca2+ via muscarinic cholinergic and beta-adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5'-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] cause release of Ca2+ from intracellular stores. The Ca2+ release evoked by cADP-ribose, but not by Ins(1,4,5)P3, was abolished by 100 microM ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca2+ signalling. When the intracellular free Ca2+ concentration ([Ca2+]i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca2+]i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.

Acetylcholine↗
PubMed1995

Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells.

The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open probability of single ATP-sensitive K(+)-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (< 5 min). The influence of repaglinide and glibenclamide on the ATP-sensitive K+ current was studied using the whole-cell patch clamp configuration. A half-maximal steady-state inhibition of the ATP-sensitive K+ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca2+ imaging on single beta TC3 cells we found that repaglinide and glibenclamide induced a concentration-dependent increase in intracellular free Ca2+ concentration ([Ca2+]i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca2+]i results from Ca2+ entry through voltage-dependent L-type Ca(2+)-channels since it is inhibited by verapamil (10 mumol/l). The effect of repaglinide and glibenclamide is partly reversible (approximately 80%).

Adenosine Triphosphate↗
PubMed1995

Role of protein kinase C in the regulation of inositol phosphate production and Ca2+ mobilization evoked by ATP and acetylcholine in rat lacrimal acini.

Stimulation of rat lacrimal acinar cells with ATP and acetylcholine (ACh) induced a rapid accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its degradation products, resulting in an initial release of Ca2+ from intracellular stores. However, after pretreating the acini with U73122 no increase in the intracellular free Ca2+ concentration ([Ca2+]i) or Ins(1,4,5)P3 production was observed. A short pretreatment with the phorbol ester 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) significantly attenuated the ATP- and ACh-induced increase in [Ca2+]i and overall inositol phosphate production. In contrast, staurosporine enhanced Ins(1,4,5)P3 and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] production and [Ca2+]i above control values in ATP- and ACh-stimulated cells. Stimulation of phospholipase C by ionomycin-evoked changes in [Ca2+]i were unaltered by pretreatment with staurosporine and PMA. The data show that a change in protein kinase C activity during cell stimulation affects the inositol phosphate metabolism and thereby the cellular Ca2+ signalling processes in lacrimal acinar cells.

Acetylcholine↗
PubMed1995

Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting beta TC3-cells by enhancement of intracellular calcium mobilization.

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.

Animals↗
PubMed1993

Ca2+ signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-trisphosphate and its role in the release of Ca2+.

The correlation between acetylcholine induced changes in the intracellular free, Ca2+ concentration ([Ca2+]i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca2+ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50-100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca2+]i and changes in the cellular Ins(1,4,5)P3 content, and it is concluded that in the individual cells Ins(1,4,5)P3 is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P3 of 2.83 x 10(-6) cm2/s (Allbritton et al., 1992, Science, 258, 1812-1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 microns where Ins(1,4,5)P3 is released in the center following a monoexponential function with a rate constant of 4 s-1. Assuming that Ins(1,4,5)P3 concentrations of 1 or 5% of the maximum value is able to release Ca2+, we calculated that Ca2+ waves can appear at a rate of 100 or 40 microns/s. The present data are consistent with Ins(1,4,5)P3 being a cellular messenger, that by diffusion, initiates the Ca2+ release from the cellular pools within the first fraction of a second.

Acetylcholine↗
PubMedDate not supplied

New insights into the regulation of glucagon secretion by glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is a potent incretin hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. One of several therapeutically important biological actions of GLP-1 in type 2 diabetic subjects is ability to induce strong suppression of glucagon secretion. The glucagonostatic action of GLP-1 results from its interaction with a specific G-protein coupled receptor resulting in the activation of adenylate cyclase and an increase in cAMP generation. In the pancreatic alpha-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion-channel activity and exocytosis of the glucagon-containing granules. In this short review, we will focus on recent advances in our understanding on the cellular mechanisms proposed to underlie the glucagonotropic action of GLP-1 and attempt to incorporate this knowledge into a working model for the control of glucagon secretion. Studies on the effects of GLP-1 on glucagon secretion are relevant to the pathogenesis of type 2 diabetes due to the likely contribution of hyperglucagonemia to impaired glucose tolerance in type 2 diabetes.

Animals↗