scholarly journals The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration

1989 ◽  
Vol 259 (3) ◽  
pp. 743-749 ◽  
Author(s):  
W S Zawalich ◽  
K C Zawalich ◽  
H Rasmussen
Keyword(s):  
Insulin Release ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Secretory Response ◽  
Second Phase ◽  
Rat Islets ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Pi Hydrolysis ◽  
Insulin Secretory Response

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.

scholarly journals Effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on islet-cell responsiveness

1991 ◽  
Vol 278 (1) ◽  
pp. 49-56 ◽  
Author(s):  
W S Zawalich ◽  
K C Zawalich ◽  
S Ganesan ◽  
R Calle ◽  
H Rasmussen
Keyword(s):  
Insulin Release ◽  
Phorbol Ester ◽  
Inositol Phosphate ◽  
Secretory Response ◽  
Second Phase ◽  
Significant Impairment ◽  
Inositol Phosphate Accumulation ◽  
Pi Hydrolysis ◽  
Quantitative Immunoblotting ◽  
Insulin Secretory Response

Collagenase-isolated rat islets were labelled for 2 h in myo-[2-3H]inositol solution supplemented with 2.75 mM-glucose. The phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 microM) was also present in some experiments. After labelling, islets were washed and then perifused in 2.75 mM-glucose to establish basal [3H]inositol-efflux and insulin-secretory rates. Subsequently, the responses of these islets to stimulation with various agonists were assessed. Inositol phosphate accumulation was measured at the termination of the perifusion. In separate experiments, the cellular location of protein kinase C (PKC) after PMA pretreatment was measured by quantitative immunoblotting of membrane and cytosolic fractions. The following observations were made. (1) Labelling in 0.1-1 microM-PMA had no deleterious effect on total [3H]inositol incorporation during the 2 h labelling period. However, islets labelled for 2 h in 1 microM-PMA were unable to respond, in terms of increases in insulin release, to a 1 microM-PMA stimulus during the subsequent perifusion. (2) As compared with the responses of control islets labelled in 2.75 mM-glucose alone, islets labelled in the additional presence of 1 microM-PMA displayed a significant impairment in phosphoinositide (PI) hydrolysis, but an enhancement of both first-and second-phase insulin secretion, in response to subsequent 20 mM-glucose stimulation. (3) Decreasing extracellular Ca2+ level to 0.1 mM and including the Ca(2+)-channel antagonist nitrendipine (0.5 microM) along with 1 microM-PMA during the [3H]inositol-labelling period did not alter the response of the islets to the subsequent addition of 20 mM-glucose. Glucose-induced PI hydrolysis was still inhibited and 20 mM-glucose-induced insulin release was still enhanced. (4) A markedly amplified and sustained insulin-secretory response to 200 microM-tolbutamide in the presence of 2.75 mM-glucose was also obtained from 1 microM-PMA-pretreated islets. This contrasts sharply with the small and transient response to tolbutamide noted in control islets. (5) When present only during the perifusion phase of the experiments, nitrendipine (0.5 microM) abolished the amplified insulin-secretory responses to both 20 mM-glucose and 200 microM-tolbutamide noted in PMA-pretreated islets. (6) Prior labelling in 1 microM-PMA dramatically amplified the insulinotropic effect of 25 mM-K+ or 5 microM-A23187 stimulation. The amplified insulin-secretory response to K+, but not to A23187, was abolished by inclusion of nitrendipine during the perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets

1988 ◽  
Vol 254 (5) ◽  
pp. E609-E616 ◽  
Author(s):  
W. S. Zawalich ◽  
V. A. Diaz ◽  
K. C. Zawalich
Keyword(s):  
High Glucose ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Metabolism ◽  
Rat Islets ◽  
Direct Measurements ◽  
Inositol Phosphate Accumulation ◽  
Pi Hydrolysis ◽  
Insulin Secretory Response

Prior exposure of isolated perifused rat islets to 20 mM glucose or 10 mM glyceraldehyde amplifies their subsequent insulin secretory response to 10 mM glucose. The involvement of phosphoinositide turnover in the induction of this "time-dependent potentiation" (TDP) was investigated. In islets in which inositol-containing phospholipids were prelabeled with myo-[2-3H]inositol, the addition of 20 mM glucose augments the efflux of 3H. This effect persists for approximately 50 min after the cessation of stimulation. Direct measurements of labeled inositol phosphate accumulation confirmed that this increase in 3H efflux is primarily the result of a persistent increase in phosphoinositide (PI) hydrolysis and not due to the slow efflux and/or degradation of performed [3H]inositol phosphates. The duration of the increase in 3H efflux parallels the duration of TDP. Mannoheptulose abolishes both the increase in 3H efflux evoked by 20 mM glucose and TDP. The omission of extracellular calcium plus 0.5 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid also abolishes both of these effects of high glucose. D-Glyceraldehyde (10 mM) addition to 3H-inositol-prelabeled islets results in an acute efflux of 3H, a persistent efflux after removal of the D-glyceraldehyde from the perifusion medium, and the induction of TDP. Similar to the results obtained with high glucose, the return of 3H efflux rates to prestimulatory values is accompanied by the abolition of TDP. These results suggest that events associated with persistent stimulant-induced increases in phosphoinositide hydrolysis may participate in the induction and maintenance of TDP.

scholarly journals A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

2004 ◽  
Vol 9 (4) ◽  
pp. 343-353 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Sarah L. Haftl ◽  
Dimitris N. Xanthos ◽  
Gregg Crumley ◽  
Mohamed Hachicha ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Column Chromatography ◽  
Large Volume ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Assay Method ◽  
Signal To Noise ◽  
Chromatography Method ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells

1986 ◽  
Vol 236 (1) ◽  
pp. 171-175 ◽  
Author(s):  
M E Monaco ◽  
R A Mufson
Keyword(s):  
Binding Affinity ◽  
Phorbol Ester ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Tumour Cells ◽  
Mammary Tumour ◽  
Phosphate Accumulation ◽  
Phosphoinositide Cycle ◽  
Inositol Phosphate Accumulation

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.

Differential effects of microtubule-altering agents on beta-cells during development

1983 ◽  
Vol 245 (4) ◽  
pp. E391-E400
Author(s):  
R. S. Hill ◽  
W. B. Rhoten
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Insulin Release ◽  
High Glucose ◽  
Deuterium Oxide ◽  
Inhibitory Effect ◽  
Secretory Response ◽  
Second Phase ◽  
Insulin Secretory Response

The effect of microtubule-altering agents on the insulin secretory response to glucose during the perinatal period was investigated with an in vitro perifusion system. Rat pancreatic mince from day 17 of gestation (D17G) to day 6 postnatally (D6PN) were perifused for 60 min in basal glucose followed by 45 min with high glucose (3.5 mg/ml) or with high glucose plus 10 mM arginine (D17G). The two phases of insulin secretion in response to high glucose developed in an age-dependent and asynchronous manner. The first phase matured between D17G and D18G, and maturation of the second phase occurred subsequently. Vinblastine (VB) (20 or 100 microM) had a differential effect on the insulin secretory response. VB did not inhibit stimulated insulin release at D17G. This absence of an inhibitory effect of VB at D17G could not be explained by the absence of polymerized tubulin because microtubules were present in the control beta-cells and, in addition, VB treatment resulted in the formation of paracrystalline deposits. Subsequently in development, and with isolated islets of the adult, VB inhibited stimulated insulin release. Heavy water (deuterium oxide, D2O) inhibited stimulated insulin secretion at D17G but blocked completely insulin release from the near-term beta-cell. The inhibition of insulin secretion by D2O was rapidly reversed when water replaced D2O in the perifusion media. The results indicate that the maturation of the second phase of insulin secretion coincides with the ability of the microtubule-altering agents to modify the insulin secretory response. One possible explanation for these findings is that at D17G the microtubules are not coupled physicochemically to other molecules or structures necessary for their role in insulin secretion to be expressed fully.

scholarly journals Anti-insulin antiserum increases inositol phosphate accumulation in rat pancreatic islets

1990 ◽  
Vol 267 (2) ◽  
pp. 339-342 ◽  
Author(s):  
E J Verspohl ◽  
H P Ammon ◽  
M Klosa
Keyword(s):  
Pancreatic Islets ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Indirect Evidence ◽  
Maximal Effect ◽  
Biologically Relevant ◽  
Rat Pancreatic Islets ◽  
Phosphate Accumulation ◽  
Control Serum ◽  
Inositol Phosphate Accumulation

The role of insulin in modulating phosphoinositide breakdown and accumulation of inositol phosphates was investigated in isolated rat pancreatic islets by using GPAIS (guinea-pig anti-insulin antiserum) that neutralizes effects of insulin in the medium. At either 3.0 mM- or 16.7 mM-glucose or 3.0 mM-glucose plus 10 microM-arecaidine propargyl ester (muscarinic receptor agonist), GPAIS (but not control serum) was able to increase InsP2 and InsP3, but not InsP, in myo-[3H] inositol-prelabelled islets. The effect of GPAIS on 3H incorporation into InsP3 was dose-dependent, with a half-maximal effect at a concentration able to bind 4004 +/- 163 microunits of insulin. A specific mass assay of the biologically relevant isomer Ins (1,4,5)P3 revealed a huge increase (greater than 3-folf). Formation of PtdIns, PtdInsP and PtdInsP2 was not affected by GPAIS. This is indirect evidence for an effect of insulin on inositide metabolism, and therefore endogenously released insulin may have led to an underestimation in earlier studies of effects of insulinotropic substances on inositol phosphate accumulation.

Accumulation of inositol phosphates in low-passage human meningioma cells following treatment with epidermal growth factor

1994 ◽  
Vol 80 (5) ◽  
pp. 890-896 ◽  
Author(s):  
Tomoki Todo ◽  
Rudolf Fahlbusch
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Human Meningioma ◽  
Phosphate Accumulation ◽  
Phospholipid Hydrolysis ◽  
Inositol Phosphate Accumulation ◽  
Epidermal Growth ◽  
Inositol Phospholipid

✓ In order to elucidate some of the signal transduction processes in human meningioma cells, the authors studied the effect of epidermal growth factor (EGF) and bromocriptine on inositol phospholipid hydrolysis, using low-passage human meningioma cells in culture. Epidermal growth factor is a well-studied mitogenic factor for meningioma cells, whereas bromocriptine is known to have an inhibitory effect on meningioma cell proliferation. The addition of EGF to meningioma cells caused stimulation of inositol phosphate accumulation in a dose-dependent manner at 60 minutes posttreatment, with the maximum effect (120% to 167% of control) achieved at a concentration of 10 ng/ml. Extraction of separate inositol phosphates revealed that inositol monophosphate (IP1) and inositol bisphosphate (IP2), but not inositol trisphosphate (IP3), accounted for the increase at 60 minutes. Kinetic analysis of EGF-stimulated inositol phospholipid hydrolysis showed that a sharp and transient increase in IP3 from 5 to 12 minutes post-EGF and a transient but more gradual increase in IP2 from 2 to 12 minutes post-EGF were followed by a gradual and steady increase in IP1, which was significantly greater than control after 5 minutes. On the other hand, long-term studies showed a down-regulation of inositol phosphate accumulation (a 64% decrease vs. control) after 7 days of treatment with EGF (10 ng/ml). Bromocriptine (5 µM) exhibited no significant effect on inositol phosphate accumulation at 60 minutes in four of five meningiomas studied. However, of two meningiomas studied with bromocriptine in combination with EGF, both showed a significant additive increase in inositol phosphate accumulation compared to those treated with EGF alone. The results suggest a close involvement of inositol phospholipid turnover in human meningioma cells in response to mitogenic stimulation by EGF.

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