Continuity and discontinuity of attachment patterns: a short-term longitudinal pilot study using a sample of late-adopted children and their adoptive mothers.

This study analysed the attachment patterns of 28 late-adopted children (placed when they were between four and seven years of age) and their adoptive mothers. The change in the children's internal working models (IWMs) within seven to eight months of their placement was evaluated. In addition, we wanted to observe the influence of a secure-autonomous maternal state of mind in facilitating the change in the children's IWMs and the possible associations between the maternal IWMs and the children's IWMs in the adoptive dyads. The separation-reunion procedure (SRP) was used for the late-adopted children in order to assess their attachment behavioural patterns, and the Manchester Child Attachment Story Task (MCAST) was used to evaluate their attachment narrative patterns. The adoptive mothers completed the Adult Attachment Interview (AAI) in order to classify their state of mind with regard to attachment. The results showed a significant change in the attachment behavioural patterns of late-adopted children, from insecure to secure (p = .002). Furthermore, the children who presented this change were predominantly placed with secure-autonomous adoptive mothers (p = .047), although the link between the adoptive mothers' representations of their attachment history and their adopted children's completed narratives was not significant. In conclusion, it seems possible to revise the attachment behaviour of late-adopted children but, for about one-third of children, the adverse history will persist at a narrative/representational level.

'Adoption and attachment theory' the attachment models of adoptive mothers and the revision of attachment patterns of their late-adopted children.

OBJECTIVE: This study examined the attachment patterns of late-adopted children (aged 4-7) and their adoptive mothers during the first 7- to 8-month period after adoption and aimed to evaluate the effect of adoptive mothers' attachment security on the revision of the attachment patterns of their late-adopted children. DESIGN: We assessed attachment patterns in 20 adoptive dyads and 12 genetically related dyads at two different times: T1 (time 1) within 2 months of adoption and T2 (time 2) 6 months after T1. METHODS: The children's behavioural attachment patterns were assessed using the Separation-Reunion Procedure and the children's representational (verbal) attachment patterns using the Manchester Child Attachment Story Task. The attachment models of the adoptive mothers were classified using the Adult Attachment Interview. RESULTS: We found that there was a significant enhancement of the late-adopted children's attachment security across the time period considered (P= 0.008). Moreover, all the late-adopted children who showed a change from insecurity to security had adoptive mothers with secure attachment models (P= 0.044). However, the matching between maternal attachment models and late-adopted children's attachment patterns (behaviours and representations) was not significant. CONCLUSIONS: Our data suggest that revision of the attachment patterns in the late-adopted children is possible but gradual, and that the adoptive mothers' attachment security makes it more likely to occur.

Glucose dose-related influence of phospholipase C and melittin on insulin release from perifused rat islets.

We studied the influence of exogenous phospholipase C (20 mU/ml) and melittin (25 micrograms/ml) on glucose-induced insulin release to further assess the role of Pl turnover as an important component in signal transduction in the B-cells. The secretory response to both substances was rapid and biphasic. However, phospholipase C did not, like melittin, elicit a secretory response in the absence of glucose. Phospholipase C elicited a half-maximal response at 4.5 mM and melittin at 2.0 mM glucose. Phospholipase C also enhanced the maximum amount of insulin release. Mannoheptulose markedly inhibited the secretory response of both melittin and phospholipase C in the presence of glucose, but the secretory effect of melittin, in the absence of glucose was not influenced. The results indicate that both melittin and phospholipase C elicit a secretory effect dependent on intact glucose metabolism. However, the variability of the effects of each agent on the glucose-dose related secretory response indicates that melittin may influence insulin release in a manner independent of nutrient metabolism and phospholipase C activity.

Effect of substitution of a permeable weak acid for the permissive role of glucose in amino acid-induced electrical activity in B-cells.

The amino acids L-leucine, L-isoleucine, and L-arginine require a subthreshold concentration of glucose to elicit insulin release and electrical activity from B-cells. There is evidence suggesting that protons couple the metabolism of glucose to the functional response of B-cells. In view of this, a permeable weak acid, sulfamerazine, was used to determine if the generation of intracellular protons could account for the permissive action of glucose. Addition of 10 mM sulfamerazine elicited constant spike activity only with 20 mM leucine. With 20 mM arginine or isoleucine, sulfamerazine induced silent depolarization no different from that caused by sulfamerazine alone. The pattern of the electrical activity of each amino acid plus 5.6 mM glucose or alpha-ketoisocaproic acid alone was qualitatively different; addition of sulfamerazine enhanced the electrical response. The permeable weak base NH4Cl at 20 mM immediately inhibited the electrical response to each amino acid plus glucose or alpha-ketoisocaproic acid alone. The effects of the permeable weak acid and base indicate that intracellular pH is important in maintaining amino acid-induced electrical activity. The permissive role of glucose may be due to provision of protons only with leucine.

Amiloride-sensitive regulation of intracellular pH in B-cells: activation by glucose.

Alterations in intracellular pH (pHi) generated by metabolism of glucose has been proposed to be a transduction device for controlling changes in K+ conductance in the plasma membrane of the B-cell leading to depolarization and cyclic variations in the membrane potential associated with spike activity. The influence of permeable weak acids or bases and amiloride inhibition of H+ extrusion by a Na:H exchanger on glucose-induced electrical activity has suggested that the electrical events are pH-sensitive. In order to document that these conditions alter pHi, we determined the influence of glucose, propionic acid, and NH4Cl, in the presence or absence of amiloride on pHi of rat islets using [14C] DMO. Glucose, 2.8 mmol/L decreased pHi by .09 unit compared to the absence of glucose (pHi = 7.08 +/- .01, M +/- SEM) and 16.7 mmol/L glucose reduced pHi by .19 unit. The glucose dose-related decrease in pHi yielded a half-maximal response at 4 mmol/L. The addition of 0.1 mmol/L amiloride had no influence on pHi without glucose and decreased pHi in the presence of 2.8 mmol/L glucose by .14 unit. The addition of 20 mmol/L propionic acid to 2.8 mmol/L glucose reduced pHi to 6.85 +/- .05, whereas 20 mmol/L NH4Cl increased pHi to 7.27 +/- .07. The addition of amiloride did not further lower the reduction in pHi elicited by 20 mmol/L propionic acid or 16.7 mmol/L glucose. These results suggest that the amiloride-sensitive Na:H exchanger plays a major role in regulation of pHi, but another modality for pHi regulation exists to compensate for inhibition of Na:H exchange under conditions of an acid load.

Phospholipase C and melittin enhancement of glucose-induced electrical activity.

The influence of exogenous phospholipase C and melittin on electrical activity in islet B cells was determined to assess the extent to which polyphosphatidylinositol turnover serves to generate components that influence the electrical events in the plasma membrane. Phospholipase C hydrolyzes polyphosphatidylinositol to diacylglycerol and polyphosphoinositol, whereas melittin increases the susceptibility of phospholipids to phospholipases and increases the permeability of the membrane to ions. Application of both 20 mU/ml phospholipase C or 0.5 mg/ml melittin to 11.1 mM glucose elicited a time-dependent enhancement of glucose-induced electrical activity that stabilized after 10 min. Phospholipase C increased both the active phase fraction and the burst frequency, whereas melittin only increased the burst frequency. These results indicate that both compounds, which disrupt the phospholipid environment of the plasma membrane, play a role in modulating the oscillatory pattern of electrical activity in the B cell, although melittin is obviously not influencing the factors controlling the ionic events in the same manner as phospholipase C.

Action of a phorbol ester on B-cells: potentiation of stimulant-induced electrical activity.

The possible role of protein kinase c in regulating the electrical events in the B-cell plasma membrane was examined by using the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a known activator of this enzyme. TPA has been found to enhance glucose- and sulfonylurea-induced insulin secretion with little or no effect on the fluxes of 86Rb+ or 45Ca2+ across the plasma membrane. TPA, 0.2 microM, did not influence the membrane potential from 0 to 5.6 mM glucose but increased by two- to threefold the fraction of the plateau phase of the oscillatory electrical activity induced by 7.0-11.1 mM glucose. This effect of TPA was completely blocked by 0.5 mM spermidine, an inhibitor of protein kinase c. However, spermidine had no influence on the electrical activity elicited by glucose alone. Glyburide, 10 nM, initiated slow depolarization and constant spike activity after about 18 and 25 min, respectively. TPA or 2.8 mM glucose reduced the lag period for glyburide to elicit an electrical response by about 75%. The duration of the spikes was increased two- to threefold by the presence of glucose or TPA with glyburide. There were also characteristic differences in the shape of the spikes under each experimental condition. Spermidine inhibited the influence of glucose, but not TPA, on the glyburide-induced electrical response. These results indicate that TPA may influence stimulant-induced electrical events via protein kinase c or by directly altering the ionic permeability of the plasma membrane.

Influence of a tumor-promoting phorbol ester on the electrical response of B-cells to glucose and glyburide.

The tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), enhances the secretory responses of pancreatic islet cells to glucose and a hypoglycemic sulfonylurea. The influence of TPA on the electrical activity induced by glucose or the sulfonylurea, glyburide, was assessed to determine whether TPA altered the conductances of the K+ and Ca2+ channels subserving depolarization and spike generation. TPA, 0.2 microM, did not alter the membrane potential obtained with 0-5.6 mM glucose. With 7.0, 8.4, and 11.0 mM glucose, TPA increased the duration of the active phase of oscillatory spike activity more than 2-fold. Upon withdrawal of glucose or glucose plus TPA, the cells hyperpolarized and spike activity ceased. In cells not exposed to TPA, the subsequent addition of 10 nM glyburide resulted in slow depolarization after 10-13 min and occurred at a rate of 1.2 +/- 0.2 mV/min. In cells preexposed to TPA, depolarization commenced by 4-8 min and occurred at a rate of 3.2 +/- 0.4 mV/min. The magnitude of depolarization was 15-20 mV with both conditions. Small amplitude spikes appeared during depolarization. After depolarization, the onset of high amplitude spikes appeared sooner in TPA-treated B-cells and occurred 2.6 times more frequently during the first 6.5 min after depolarization than in B-cells not exposed to TPA. A stable pattern of spike generation was achieved twice as fast in TPA-treated cells. TPA enhancement of glucose- and glyburide-induced spike activity suggests that the insulinotropic action of TPA is mediated by augmenting the influx of Ca2+ into the B-cell via voltage-sensitive Ca2+ channels.

Role of pH as a transduction device in triggering electrical and secretory responses in islet B cells.

Changes in pH alter the oscillatory pattern of glucose-induced electrical activity of mouse islet B cells, thereby supporting the hypothesis that changes in intracellular pH (pHi) resulting from glucose metabolism serve as a coupling factor between metabolic and cationic events. A decrease in pHi in the present of 11.1 mM glucose induces an increase in the duration of the active phase similar to that evoked by higher concentrations of glucose. Regulation of pHi appears to occur by Na:H and HCO3:Cl exchange in the plasma membrane, because inhibition by 0.1 mM amiloride and 0.5 mM 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS), respectively, induces constant spike activity in the presence of 11.1 mM glucose. If the pH coupling hypothesis is correct, then inhibition of the putative pH regulatory mechanisms and the subsequent decrease in pHi should elicit electrical activity in the presence of subthreshold glucose (less than 7.0 mM). Amiloride induced electrical activity (threshold) at 4.4 +/- 0.3 mM (mean +/- SEM) glucose. The threshold for DIDS was 5.4 +/- 0.2 glucose, whereas with glucose alone the threshold was achieved at 7.0 +/- 0.4 mM. Thus the generation of H+ by glucose may trigger changes in ionic conductances that induce the typical electrical response. Amiloride was found to elicit a secretory response at subthreshold glucose (4.2-7.0 mM) in perifused rat islets. This indicates that pH-induced changes in the ionic events in the B cells also play an important role in information transfer to the secretory complex.

Hyperosmolarity and cardiac function in chronic diabetic rat heart.

Serum hyperosmolarity is commonly associated with the poorly controlled diabetic state. The current investigation revealed that increased perfusate osmolarity using either glucose or mannitol caused a gradual decline in the aortic output of hearts from both normal and diabetic rats. However, aortic output decreased less rapidly in hearts from diabetic rats. Decreasing the extracellular calcium concentration to a level resulting in one-third of the maximal aortic output elicited a greater and more prolonged increase in aortic output from the diabetic compared with the normal heart when perfusate osmolarity was progressively increased. The coronary conductance of diabetic rat hearts improved when hyperosmolar solutions of either mannitol or glucose were utilized, irrespective of the external calcium ion concentration. In normal hearts, the coronary conductance tended to decline at higher perfusate osmolarities. The increased coronary conductance may play a role in the enhanced performance of diabetic hearts at higher levels of osmolarity. Hyperosmolar solutions influence the performance of hearts from chronic diabetic rats in a positive inotropic manner at low extracellular calcium concentrations (1 mM). These lower calcium levels are close to the normal physiological range of the freely ionized serum calcium. This effect is greater in hearts from diabetic animals than from normal animals and is similar to that obtained when perfusate calcium is increased. The results suggest that hyperosmolar solutions enhance calcium availability in the hearts of diabetic animals.

Altered sensitivity of chronic diabetic rat heart to calcium.

An alteration in calcium metabolism in cardiac muscle was observed in diabetic rats 3 mo after streptozotocin treatment. Depression of cardiac output and left ventricular pressure development were more sensitive to decreased extra-cellular calcium in hearts from diabetic than from control animals and occurred within the normal physiological range of freely ionized serum calcium. This decrease in calcium sensitivity was not present after 2 wk of diabetes. In vivo treatment with insulin for 1 mo completely reversed the effect. Addition of octanoate (0.3 mM) to the perfusate of isolated hearts completely reversed the defect, whereas epinephrine (25 nM) only partially reversed it. When the glucose concentration of the perfusate was decreased, the function of diabetic hearts declined and was further diminished at decreasing calcium levels. Hearts from normal rats were unaffected. These results suggest that there is a defect in calcium metabolism or flux in the chronic diabetic rat heart.

The role of chemiosmotic lysis in the exocytotic release of insulin.

The role of chemiosmotic lysis in the exocytotic release of insulin has been studied using perifused rat pancreatic islets of Langerhans. Established criteria for osmotic lysis of secretory granules requires proton translocation across the secretory granule membrane and the influx of a permeant anion. The consequent increase in granule osmolarity induces water entry and granule lysis. A proton gradient has been previously established to exist across the insulin secretory granule membrane. We have examined the sensitivity of insulin release to 1) hyperosmolar solutions, 2) replacement of medium Cl-, 3) replacement of medium Na+, and 4) anion transport inhibitors. The addition of 200-600 mM sucrose resulted in a 32-69% inhibition of insulin release due to 16.7 mM glucose. Replacement of Cl- by isethionate or SO4--reversibly inhibited glucose-induced insulin release by 47% and 78%, respectively. Na+ replacement by choline did not influence the secretory response. 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid (500 microM) and probenecid (10 mM) inhibited insulin release by 73% and 79%, respectively. These drugs are known to inhibit anion exchange in erythrocytes and may be influencing Cl- entry into the secretory granule fused to the plasma membrane by a similar mechanism. Furosemide inhibits NaKCl2 cotransport in erythrocytes, but had no influence on glucose-induced insulin release, suggesting that Cl- does not enter the secretory granule by this pathway. The primary criteria for the participation of a chemiosmotic mechanism subserving lysis of the insulin secretory granule are fulfilled by these results.

Endogenous prostaglandin synthesis and glucose-induced insulin secretion from the adult rat pancreatic islet.

Although exogenous prostaglandins are recognized modulators of insulin secretion, the relationship between their endogenous synthesis and insulin secretion has not been rigorously studied in isolated adult rat islets. Using 3H-arachidonic acid as a tracer, we evaluated the effect of glucose stimulation upon the incorporation of this fatty acid into islet phospholipids and prostaglandins (separated by extraction and sequential silicic acid, thin-layer and paper chromatography). We observed that 3H-arachidonic acid was incorporated into islet phospholipids and prostaglandins under basal conditions (0.3 mg/ml glucose). Furthermore, exposure of islets to a stimulatory glucose concentration led to significant increases in the recovery of 3H-arachidonic acid-derived radioactivity in islet phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylinositol as well as into all of the measured prostaglandins (A2, B2, D2, E2, and F2 alpha). The most marked increases in recovered radioactivity resulting from a stimulatory glucose concentration were in islet phosphatidylethanolamine and prostaglandin A2 (which we believe to be derived, in large part, from endogenously synthesized prostaglandin E2). These glucose-induced increases in 3H-arachidonic acid-derived radioactivity in both the phospholipid and the prostaglandin fractions were eliminated by the inhibition of phospholipase A2 activity with mepacrine or by the inhibition of cyclooxygenase activity with sodium salicylate. When islets prelabeled with 3H-arachidonic acid were exposed to a high glucose concentration in a perifusion system, there was a brisk extracellular release of radioactivity (presumably representing unidentified prostaglandins) that began within 1 min and that peaked slightly before the peak of the first phase of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

pH modulation of glucose-induced electrical activity in B-cells: involvement of Na/H and HCO3/Cl antiporters.

Regulation of intracellular pH is an essential function and may be especially significant in the B-cell in which the influence of glucose on electrical activity is modulated by alterations in pH. Two possible regulatory processes have been examined: Na/H and HCO3/Cl exchange, by using inhibitors, an ionophore, and changes of ionic concentrations. In the presence of 11.1 mM glucose we found that DIDS, an inhibitor of anion exchange, elicited a dose-response increase in the relative duration of the active phase with an ED50 of 99 microM. Probenecid (0.5 mM), an inhibitor of anion fluxes, also augmented the electrical activity (EA) due to glucose. Withdrawal of HCO-3 elicited constant spike activity followed by a resumption of burst activity with a greater duration of the active phase compared to control. These data are consistent with predicted cellular acidification. However, reduction of Cl-o by isethionate substitution produced no marked effect on EA. In contrast, SO-4- substitution for Cl- resulted in variable effects characterized by constant spike activity or a decrease in the duration of the active and silent phases along with silent hyperpolarization. Tributyltin, a Cl/OH, ionophore enhanced EA at 0.25 microM with 120 mM Cl-o, but reduced EA with 10 mM Cl- as would be predicted with either cellular acidification or alkalinization, respectively. Amiloride at 100 microM elicited constant spike activity perhaps due to inhibition of Na/H exchange. Reduction of Na+o from 142.8 to 40.8 mM had a similar effect and enhanced the influence of amiloride. It appears therefore that interference with putative pH regulatory mechanisms in the B-cell are consistent with the hypothesis that cell pH is involved in regulation of EA.

Modification of glucose-induced insulin release by alteration of pH.

Protons (H+) generated by glucose metabolism have been proposed to serve as a coupling factor between cationic and secretory events in the B-cell. We have examined the influence of alteration of extracellular or intracellular pH (pHo or pHi) on dynamic secretory responses of perfused rat islets to 4.2, 8.4, or 16.7 mM glucose. Reduction of pHo from 7.4 to 7.0 inhibited the secretory response to 16.7, but not 8.4 mM glucose, by 47% during the 30-min period following medium change. Increase of pHo from 7.4 to 7.8 had no influence on the secretory response to glucose. Alteration of pHo had no influence on basal insulin release in the presence of 4.2 mM glucose. Sulfamerazine (5 mM), a permeable weak acid, augmented the secretory response to 8.4 mM glucose by 60% but had no influence on the response to 16.7 mM glucose. In contrast, imidazole (10 mM), a permeable weak base, inhibited the secretory response to both 8.4 (62%) and 16.7 mM (72%) glucose. Another weak base, NH4Cl (20 mM), also inhibited the secretory response to 8.4 (61%) and 16.7 mM (68%) glucose. Alteration of pHi by sulfamerazine and imidazole did not alter basal insulin release in the presence of 4.2 mM glucose. A comparison of the present findings to those obtained for the influence of pH on glucose-induced electrical activity indicates that alteration of pHi, and not pHo, induces parallel effects on glucose-induced electrical and secretory events.

Stimulus-secretion coupling in beta-cells: modulation by pH.

We have examined the influence of changes in pH on the oscillatory pattern of electrical activity (EA) in the beta-cell by altering medium pH (pHo) and using permeable weak buffers to alter intracellular pH (pHi). A decrease in pH in the presence of glucose elicited depolarization to the active phase and constant spike activity, whereas an increase in pH elicited a decrease in spike activity or silent hyperpolarization. On inhibition of HCO3:Cl antiport by addition of DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), probenecid, or withdrawal of medium HCO-3, there was an increase in the duration of the active phase. A similar result was obtained on the inhibition of Na:H antiport by the addition of amiloride or the reduction of medium [Na+]. The influence of H+ and glucose has been proposed to decrease K+ permeability (PK). However, the influence of pH on 86Rb+ efflux was most effective at subthreshold or 4.2 mM glucose; only a moderate decrease in PK occurred at 8.3 mM glucose, and no effect was obtained at 16.7 mM glucose. Alteration of pHi, and not pHo, induces similar effects on glucose-induced electrical and secretory events. There is a clear dissociation between the influence of inhibitors of the Na:H and HCO3:Cl antiporters on the electrical and secretory events. DIDS and amiloride increased glucose-induced EA, but markedly inhibited the secretory response to glucose. It is evident that pH modulates the electrical events and cationic fluxes and ultimately influences the transduction of information to the mechanisms controlling the secretory process in the beta-cell.

Influence on anion transport on glucose-induced electrical activity in the B-cell.

We have previously shown that the effect of glucose on electrical activity (EA) in islet B-cells is altered by modification of pH. The regulation of intracellular pH (pHi) in nerve and muscle cells is coupled to anion exchange. In the present study we have examined the involvement of HCO3:Cl exchange across the plasma membrane in the maintenance of glucose-induced EA in B-cells. 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS), an inhibitor of anion exchange, elicited a dose-related stimulation of EA in the presence of 11.1 mM glucose. The increase in the relative duration of the active phase (constant spike activity) was first observed at 20 microM DIDS, and a nearly maximal effect was obtained at 200 microM. The substitution of HCO3- by a Hepes buffer elicited constant spike activity. The application of 0.25 microM tributyltin, an electroneutral Cl:OH exchanger, also enhanced EA as indicated by an increase in the duration of the active phase. The influence of HCO3- withdrawal, DIDS, and tributyltin all elicited electrical events similar to that obtained by a decrease in pHi. Our results suggest that anion exchange may be involved in the regulation of electrical events in the B-cell by influencing pHi, as has been documented to occur in invertebrate nerve and muscle.

Glucose-induced proton uptake in secretory granules of beta-cells in monolayer culture.

The weak base acridine orange (AO) has been shown to be accumulated by the insulin-containing secretory granules of cultured beta-cells in response to high glucose. Various lines of evidence indicate that this accumulation is due to a pH gradient. Thus ionophores such as monensin and nigericin abolish the glucose-induced accumulation, and a high concentration of the weak base, benzylamine, results in swelling of the granules. In the absence of glucose, ATP addition to digitonin-permeabilized cells also results in dye uptake. These data also suggest that a primary or secondary active accumulation mechanism for hydrogen ions exists across the granule membrane.

Glucose-induced electrical activity in pancreatic beta-cell: modulation by pH.

Studies have been undertaken to examine the influence of changes in pH on the electrical activity in mouse islet beta-cells. Extracellular acidification or the presence of the permeable weak acid glycodiazine was found to alter the cyclic nature of glucose-induced electrical activity leading to depolarization and constant spike activity. Conversely, the presence of the permeable weak base imidazole was found to transiently lead to hyperpolarization with concomitant inhibition of electrical activity. Monensin, an electroneutral Na:H antiporter, was found to inhibit glucose-induced electrical activity, even in the presence of glycodiazine. These results suggest that protons may play an important role in the regulation and/or generation of the oscillatory pattern of electrical activity in the beta-cell.