SAUNA QB - Array: The realization of a new concept in radioxenon detection.

We introduce a new concept in radioxenon detection - the radioxenon Array, defined as a system where air sampling and activity measurement is performed at multiple locations, using measurement units that are less sensitive, but on the other hand less costly, and easier to install and operate, compared to current state-of-the-art radioxenon systems. The inter-unit distance in the Array is typically hundreds of kilometres. Using synthetic nuclear explosions together with a parametrized measurement system model, we argue that, when such measurement units are combined into an Array, the aggregated verification performance (detection, location, and characterization) can be high. The concept has been realized by developing a measurement unit named SAUNA QB, and the world's first radioxenon Array is now operating in Sweden. The operational principles and performance of the SAUNA QB and the Array is described, and examples of first measured data are presented, indicating a measurement performance according to expectations.

Somatostatin promotes glucose generation of Ca2+oscillations in pancreatic islets both in the absence and presence of tolbutamide.

Many cellular processes, including pulsatile release of insulin, are triggered by increase of cytoplasmic Ca2+. This study examines how somatostatin affects glucose generation of cytoplasmic Ca2+ oscillations in mouse islets in absence and presence of tolbutamide blockade of the KATP channels. Ca2+ was measured with dual wavelength microflurometry in isolated islets loaded with the indicator Fura-2. Rise of glucose from 3 to 20 mM evoked introductory lowering of Ca2+ prolonged by activation of somatostatin receptors. During continued superfusion exposure to somatostatin triggered oscillations mediated by periodic increase from the basal level (absence of tolbutamide) or by periodic interruption of an elevated level (presence of tolbutamide). In the latter situation the oscillations were transformed into sustained elevation by activation of muscarinic receptors (acetylcholine) or increase of cyclic AMP (IBMX, 8-bromo-cyclic AMP, forskolin). The observed effect of cyclic AMP raises the question whether high proportions of the glucagon-producing α-cells promote steady-state elevation of Ca2+. In support for this idea somatostatin was found to trigger glucose-induced Ca2+ oscillations essentially in small islets that contain very few α-cells. The results indicate that somatostatin promotes glucose generation of Ca2+oscillations with similar characteristics both in the absence and presence of functional KATP channels.

Sulfonylurea Blockade of KATP Channels Unmasks a Distinct Type of Glucose-Induced Ca2+ Decrease in Pancreatic ß-Cells.

OBJECTIVES: This study aimed to explore how sulfonylurea blockade of KATP channels affects the early Ca signals for glucose generation of insulin release. METHODS: Cytoplasmic Ca was measured with ratiometric microfluorometry in isolated mouse islets loaded with Fura-PE3. RESULTS: After sulfonylurea blockade of the KATP channels (50 µM-1 mM tolbutamide or 1 µM-1 mM gliclazide), increase of glucose from 3 to 20 mM resulted in suppression of elevated Ca during a 3- to 5-minute period. The Ca decrease was shorter after inhibition of the Na/K pump with ouabain (10 and 100 µM) but prolonged when the α2A adrenoceptors were activated with clonidine (1 and 10 nM) or epinephrine (10 nM). Inhibition of the sarco/endoplasmic reticulum Ca-ATPase pump with 10 µM cyclopiazonic acid counteracted the action of 10 nM clonidine, making the Ca decrease shorter than in controls. Extended superfusion of islets with a medium containing 20 mM glucose and 1 mM tolbutamide sometimes resulted in delayed appearance of Ca oscillations mediated by periodic interruption of elevated Ca. CONCLUSIONS: Increase of glucose generates prompt suppression of cytoplasmic Ca in ß-cells lacking functional KATP channels. Activation of α2A adrenoceptors markedly prolongs the period of glucose-induced Ca decrease, an effect counteracted by cyclopiazonic acid.

Small Mouse Islets Are Deficient in Glucagon-Producing Alpha Cells but Rich in Somatostatin-Secreting Delta Cells.

Small and big mouse islets were compared with special reference to their content of glucagon-producing α-cells and somatostatin-producing δ-cells. Areas stained for glucagon and somatostatin were measured in the largest cross section of small (diameter < 60 µm) and big (diameter > 100 µm) islets. Comparison of the areas indicated proportionally more δ- than α-cells in the small islets. After isolation with collagenase these islets were practically devoid of α-cells. We evaluated the functional importance of the islet size by measuring the Ca(2+) signal for insulin release. A majority of the small islets responded to the hyperpolarization action of somatostatin with periodic decrease of cytoplasmic Ca(2+) when glucose was elevated after tolbutamide blockade of the KATP channels.

Activation of alpha adrenergic and muscarinic receptors modifies early glucose suppression of cytoplasmic Ca(2+) in pancreatic ß-cells.

Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca(2+) ([Ca(2+)]i) below baseline in pancreatic ß-cells. The period of [Ca(2+)]i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α2A and muscarinic M3 receptors affects the initial [Ca(2+)]i response to increase of glucose from 3 to 20mM in ß-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca(2+)]i during 90-120 s followed by rise due to opening of voltage-dependent Ca(2+) channels. The period of [Ca(2+)]i decrease was prolonged by activation of the α2A adrenergic receptors (1 µM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M3 receptors (0.1 µM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10-100 µM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca(2+)]i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca(2+)]i regulates the subsequent ß-cell response to glucose.

Isolated mouse islets respond to glucose with an initial peak of glucagon release followed by pulses of insulin and somatostatin in antisynchrony with glucagon.

Recent studies of isolated human islets have shown that glucose induces hormone release with repetitive pulses of insulin and somatostatin in antisynchrony with those of glucagon. Since the mouse is the most important animal model we studied the temporal relation between hormones released from mouse islets. Batches of 5-10 islets were perifused and the hormones measured with radioimmunoassay in 30s fractions. At 3mM glucose, hormone secretion was stable with no detectable pulses of glucagon, insulin or somatostatin. Increase of glucose to 20mM resulted in an early secretory phase with a glucagon peak followed by peaks of insulin and somatostatin. Subsequent hormone secretion was pulsatile with a periodicity of 5min. Cross-correlation analyses showed that the glucagon pulses were antisynchronous to those of insulin and somatostatin. In contrast to the marked stimulation of insulin and somatostatin secretion, the pulsatility resulted in inhibition of overall glucagon release. The cytoarchitecture of mouse islets differs from that of human islets, which may affect the interactions between the hormone-producing cells. Although indicating that paracrine regulation is important for the characteristic patterns of pulsatile hormone secretion, the mouse data mimic those of human islets with more than 20-fold variations of the insulin/glucagon ratio. The data indicate that the mouse serves as an appropriate animal model for studying the temporal relation between the islet hormones controlling glucose production in the liver.

The neurotransmitter ATP triggers Ca2+ responses promoting coordination of pancreatic islet oscillations.

OBJECTIVES: Pulsatile insulin release into the portal vein is critically dependent on entrainment of the islets in the pancreas into a common oscillatory phase. Because the pulses reflect periodic variations of the cytoplasmic Ca concentration ([Ca]i), we studied whether the neurotransmitters adenosine triphosphate (ATP) and acetylcholine promote synchronization of [Ca]i oscillations between islets lacking contact. METHODS: Medium-sized and small mouse islets and cell aggregates were used for measuring [Ca]i with the indicator fura-2. RESULTS: Exposure to acetylcholine resulted in an initial [Ca]i peak followed by disappearance of the [Ca]i oscillations induced by 11-mmol/L glucose. The effect of ATP was often restricted to an elusive [Ca]i peak. The incidence of distinct [Ca]i responses to ATP increased under conditions (accelerated superfusion, small islets, or cell aggregates) intended to counteract purinoceptor desensitization owing to intercellular accumulation of ATP. Attempts to imitate neural activity by brief (15 seconds) exposure to ATP or acetylcholine resulted in temporary synchronization of the glucose-induced [Ca]i oscillations between islets lacking contact. CONCLUSIONS: The data support the idea that purinergic signaling has a key role for coordinating the oscillatory activity of the islets in the pancreas, reinforcing previous arguments for the involvement of nonadrenergic, noncholinergic neurons.

Pulsatility of insulin release--a clinically important phenomenon.

The mechanisms and clinical importance of pulsatile insulin release are presented against the background of more than half a century of companionship with the islets of Langerhans. The insulin-secreting beta-cells are oscillators with intrinsic variations of cytoplasmic ATP and Ca(2+). Within the islets the beta-cells are mutually entrained into a common rhythm by gap junctions and diffusible factors (ATP). Synchronization of the different islets in the pancreas is supposed to be due to adjustment of the oscillations to the same phase by neural output of acetylcholine and ATP. Studies of hormone secretion from the perfused pancreas of rats and mice revealed that glucose induces pulses of glucagon anti-synchronous with pulses of insulin and somatostatin. The anti-synchrony may result from a paracrine action of somatostatin on the glucagon-producing alpha-cells. Purinoceptors have a key function for pulsatile release of islet hormones. It was possible to remove the glucagon and somatostatin pulses with maintenance of those of insulin with an inhibitor of the P2Y(1) receptors. Knock-out of the adenosine A(1) receptor prolonged the pulses of glucagon and somatostatin without affecting the duration of the insulin pulses. Studies of isolated human islets indicate similar relations between pulses of insulin, glucagon, and somatostatin as found during perfusion of the rodent pancreas. The observation of reversed cycles of insulin and glucagon adds to the understanding how the islets regulate hepatic glucose production. Current protocols for pulsatile intravenous infusion therapy (PIVIT) should be modified to mimic the anti-synchrony between insulin and glucagon normally seen in the portal blood.

Glucose generates coincident insulin and somatostatin pulses and antisynchronous glucagon pulses from human pancreatic islets.

The kinetics of insulin, glucagon and somatostatin release was studied in human pancreatic islets. Batches of 10-15 islets were perifused and the hormones measured with RIA in 30-sec fractions. Increase of glucose from 3 to 20 mm resulted in a brief pulse of glucagon coinciding with suppression of basal insulin and somatostatin release. There was a subsequent drop of glucagon release concomitant with the appearance of a pronounced pulse of insulin and a slightly delayed pulse of somatostatin. Continued exposure to 20 mm glucose generated pulsatile release of the three hormones with 7- to 8-min periods accounting for 60-70% of the secreted amounts. Glucose caused pronounced stimulation of average insulin and somatostatin release. However, the nadirs between the glucagon pulses were lower than the secretion at 3 mm glucose, resulting in 18% suppression of average release. The repetitive glucagon pulses were antisynchronous to coincident pulses of insulin and somatostatin. The resulting greater than 20-fold variations of the insulin to glucagon ratio might be essential for minute-to-minute regulation of the hepatic glucose production.

Absence of adenosine A1 receptors unmasks pulses of insulin release and prolongs those of glucagon and somatostatin.

AIMS: Extracellular ATP modulates pulsatile release of insulin, glucagon and somatostatin by activating P2Y(1) receptors. The present study examines if adenosine via A(1) receptors (A(1)R) interferes with pulsatile islet hormone release. MAIN METHODS: Pancreas was perfused in mice expressing or lacking the A(1) receptor and the hormones measured with radioimmunoassay. Cytoplasmic Ca(2+) was recorded in isolated beta-cells using the fura-2 indicator. KEY FINDINGS: Addition of 10 microM adenosine removed the Ca(2+) transients supposed to coordinate the insulin release pulses. This effect of adenosine was counteracted by 100 nM of the A(1)R antagonist DPCPX. In situ perfusion of the pancreas indicated two phases of islet hormone release when glucose was raised from 3.3 to 16.7 mM. The first phase was characterized by a brief dip followed by a peak, which was more pronounced for insulin and somatostatin than for glucagon. The second phase was markedly affected by knock out of A(1)R. The wild-type A(1)R (+/+) mice, usually lacked statistically verified insulin pulses but generated antisynchronous glucagon and somatostatin pulses with half-widths of 4 min. In the A(1)R (-/-) mice time-average release of insulin during the second phase was almost three times higher than in the controls and 30% of the hormone was released as distinct pulses with half-widths of 3 min. The absence of the A(1)R receptor resulted in 50% prolongation of the pulse cycles of glucagon and somatostatin and loss of their antisynchronous relationship. SIGNIFICANCE: The A(1)R receptor is important both for the amplitude (insulin) and duration (glucagon and somatostatin) of islet hormone pulses.

Effects of external ATP on Ca(2+) signalling in endothelial cells isolated from mouse islets.

External ATP is believed to initiate and propagate Ca(2+) signals co-ordinating the insulin release pulses within and among the different islets in the pancreas. The possibility that islet endothelial cells participate in this process was evaluated by comparing the effects on [Ca(2+)](i) of purinoceptor activation in these cells with those in beta-cells. beta-Cell-rich pancreatic islets were isolated from ob/ob mice and dispersed into single cells/aggregates. After culture with or without endothelial cell growth supplement (ECGS), the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was measured with ratiometric fura-2 technique. Presence of ECGS or prolongation of culture (>5 days) resulted in proliferation of endothelial cells and altered their phenotype from rounded to elongated. Endothelial cells, preliminarily identified by attachment of Dynabeads coated with the Bandeiraea simplicifolia 1 lectin (BS-1), responded in a similar way as those stained with CD31 antibodies after measurements of [Ca(2+)](i). Spontaneous transients and oscillations of [Ca(2+)](i )were seen in beta-cells, but not in endothelial cells exposed to 20 mM glucose. Addition of ATP (10 microM) resulted in pronounced and more extended rise of [Ca(2+)](i) in endothelial cells than in beta-cells. The endothelial cells differed from the beta-cells by also responding with a rise of [Ca(2+)](i) to 10 microM UTP, but not to equimolar ADP and acetylcholine. The results support the idea of mutual interactions between islet endothelium and beta-cells based on ATP-induced Ca(2+) signals. It is suggested that the endothelial cells have a tonic inhibitory action on beta-cell P2 purinoceptors resulting in impaired synchronization of the insulin release pulses.

Pulses of somatostatin release are slightly delayed compared with insulin and antisynchronous to glucagon.

It was early proposed that somatostatin-producing delta-cells in pancreatic islets have local inhibitory effects on the release of insulin and glucagon. Recent observations that pulses of insulin and glucagon are antisynchronous make it important to examine the temporal characteristics of glucose-induced somatostatin release. Analysis of 30 s fractions from the perfused rat pancreas indicated that increase of glucose from 3 to 20 mmol/l results in initial suppression of somatostatin release followed by regular 4-5 min pulses. During continued exposure to 20 mmol/l glucose, the pulses of somatostatin overlapped those of insulin with a delay of 30 s. Somatostatin and glucagon pulses were coupled in antisynchronous fashion (phase shift 2.4+/-0.2 min), supporting the idea that the delta-cells have a local inhibitory effect on glucagon release. It was possible to remove the pulses of somatostatin and glucagon with maintenance of the insulin rhythmicity by addition of 1 micromol/l of the P2Y(1) receptor antagonist MRS 2179.

Phospholipase A2 is important for glucose induction of rhythmic Ca2+ signals in pancreatic beta cells.

OBJECTIVES: Pancreatic beta cells respond to glucose stimulation with pulses of insulin release generated by oscillatory rises of the cytoplasmic Ca2+ concentration ([Ca2+]i). The observation that exposure to external ATP and other activators of cytoplasmic phospholipase A2 (cPLA2) rapidly induces rises of [Ca2+]i similar to ordinary oscillations made it important to analyze whether suppression of the cPLA2 activity affects glucose-induced [Ca2+]i rhythmicity in pancreatic beta cells. METHODS: Ratiometric fura-2 technique was used for measuring [Ca2+]i in single beta cells and small aggregates prepared from ob/ob mouse islets. RESULTS: Testing the effects of different inhibitors of cPLA2 in the presence of 20 mM glucose, it was found that N-(p-amylcinnamoyl)anthranilic acid (ACA) removed the oscillations at a concentration of 25 microM, arachidonyl trifluoromethyl ketone (AACOCF3) at 10 microM, and bromoenol lactone (BEL) at 10 to 15 microM. Withdrawal of ACA and BEL resulted in reappearance of the oscillations. Suppression of the arachidonic acid production by addition of 5 microM of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267) effectively removed the [Ca2+]i oscillations, an effect reversed by removal of the inhibitor or addition of 100 microM tolbutamide. Suppression of the arachidonic acid production had a restrictive influence also on the transients of [Ca2+]i supposed to synchronize the beta-cell rhythmicity. Although less sensitive than the oscillations, most transients disappeared during exposure to 50 microM ACA or 35 microM RHC 80267. CONCLUSIONS: The results support the idea that cyclic variations of cPLA2 activity are important for the generation and synchronization of the beta-cell [Ca2+]i oscillations responsible for pulsatile release of insulin.

Glucose induces glucagon release pulses antisynchronous with insulin and sensitive to purinoceptor inhibition.

Both increase of the glucose concentration and activation of purinoceptors are known to affect pancreatic alpha-cells. Effects obtained with various purino derivatives at 2.8 and 8.3 mmol/liter glucose have been taken to indicate that external ATP is less potent than adenosine as a stimulator of glucagon release. However, when making a corresponding comparison at 20 mmol/liter glucose, we observed marked stimulation of glucagon release from isolated rat islets with 100 micromol/liter adenosine-5-O-2-thiodiphosphate but inhibition with 10 micromol/liter adenosine. Analyses of 30-sec samples of perfusate from rat pancreas indicated that a rise of the glucose concentration from 3 to 20 mmol/liter rapidly induces a glucagon peak followed by regular 4- to 5-min pulses. The glucagon pulses preceded those of insulin with a phase shift (1.8 +/- 0.1 min) near half the interpeak interval. Because of the antisynchrony, the maximal glucagon effect on liver cells will be manifested during periods with low concentrations of insulin. In support for the idea that neural P2Y(1) receptors are important for coordinating the secretory activity of the islets, both the insulin and glucagon pulses disappeared in the presence of the purinoceptor inhibitor MRS 2179 (10 micromol/liter). However, in contrast to what was observed for insulin, MRS 2179 lowered average glucagon release to the level of the oscillatory nadirs.

Inhibition of purinoceptors amplifies glucose-stimulated insulin release with removal of its pulsatility.

External ATP has been proposed to be an autocrine regulator of glucose-stimulated insulin secretion and responsible for the synchronization of the Ca2+ rhythmicity in the beta-cells required for a pulsatile release of insulin from the pancreas. The importance of external ATP for glucose-stimulated insulin release was evaluated in rats with the aid of 2-deoxy-N-methyladenosine-3,5-bisphosphate (MRS 2179), an inhibitor of the purinoceptors known to affect the Ca2+ signaling in beta-cells. The concentration of cytoplasmic Ca2+ was measured in single beta-cells and small aggregates with ratiometric fura-2 technique and the release of insulin recorded from isolated islets and the perfused pancreas. Addition of 1 micromol/l ATP induced premature cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations similar to those found in beta-cells exposed to 20 mmol/l glucose. In most experiments, the presence of 10 micromol/l MRS 2179 did not remove the glucose-induced [Ca2+]i rhythmicity in single beta-cells or the synchronization seen in coupled cells. Nevertheless, the same concentration of MRS 2179 promptly interrupted the pulsatility (frequency 0.22 +/- 0.01/min) of insulin secretion, raising the total amounts released from the pancreas. Prolonged exposure of islets to 1 and 10 micromol/l MRS 2179 enhanced insulin secretion at 20 mmol/l glucose 33% (P < 0.05) and 63% (P < 0.01), respectively, without affecting the release at 3 mmol/l glucose. The results support the idea that neural ATP signals entrain the islets into a common rhythm resulting in pulsatile release of insulin and that glucose stimulation of the secretory activity is counteracted by accumulation of inhibitory ATP around the beta-cells.

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations.

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.

Pancreatic beta-cells communicate via intermittent release of ATP.

The role of external ATP for intercellular communication was studied in glucose-stimulated pancreatic beta-cells isolated from ob/ob mice. Digital image analyses with fura-2 revealed spontaneous transients of cytoplasmic Ca2+ appearing in synchrony in the absence of cell contacts. After removal of slow oscillations with methoxyverapamil, addition of ATP (0.1-100 microM) resulted in prompt firing of a transient, followed by suppression of the generation and synchronization of spontaneously occurring transients. It was possible to trigger transients during the suppressive phase by raising the concentration of ATP. The dual action of ATP was mimicked by ADP or 2-methylthio-ATP but not by AMP or UTP. The number of spontaneous transients and their synchronization were reduced in the presence of the dephosphorylating agent apyrase. Additional evidence that intermittent release of ATP participates in the generation of spontaneous Ca2+ transients was obtained from the suppression observed from use of antagonists of the purinoceptors [suramin (0.3-30 microM), pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10-30 microM) and 2-deoxy-N-methyladenosine (MRS 2179; 0.3-30 microM)] or from counteracting beta-cell release of ATP by inhibiting exocytosis with 100 nM epinephrine, 100 nM somatostatin, or lowering the temperature below 30 degrees C. The data indicate that ATP has time-dependent actions (prompt stimulation followed by inhibition) on the generation of Ca2+ transients mediated by P2Y receptors. It is proposed that beta-cells both receive a neural ATP signal with coordinating effects on their Ca2+ oscillations and propagate this message to adjacent cells via intermittent release of ATP combined with gap junction coupling.

Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic beta-cells.

A key question for understanding the mechanisms of pulsatile insulin release is how the underlying beta-cell oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the beta-cells by precipitating transients of [Ca2+]i. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol.min-1.mg protein-1) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 microM) and in a 16 times higher frequency of [Ca2+]i transients in their beta-cells. Hemin (0.1 and 1.0 microM), the natural substrate for HO, promoted the appearance of [Ca2+]i transients, and 10 microM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating Ca2+ signals with coordinating effects on the beta-cell rhythmicity.

Stretch activation of Ca2+ transients in pancreatic beta cells by mobilization of intracellular stores.

INTRODUCTION: Nonadrenergic, noncholinergic neurons have been proposed to synchronize pulsatile insulin release from the islets in the pancreas by triggering transient increases of the cytoplasmic Ca2+ concentration ([Ca2+]i) in beta-cells via an inositol trisphoshate-dependent mechanism. AIMS: To test whether pancreatic beta-cells respond to stretch activation with similar types of transients and whether these Ca signals propagate to other beta-cells in the presence and absence of cell contacts. METHODOLOGY: Single cells and small aggregates were prepared from beta-cell-rich islets from mice. After 2-5 days of culture, [Ca2+]i was measured with digital imaging and the indicator fura-2 during superfusion with a medium containing 20 mmol/L glucose and 50 micromol/L methoxyverapamil. Membrane stretch was induced by osmotic swelling or focal touch stimulation. RESULTS: Lowering the medium osmolarity with 100-102 mOSM/L by removal of sucrose or by dilution resulted in a 2-3-fold increase in the number of transients during an initial 5-minute period. Sucrose omission was stimulatory also after isosmolar replacement with readily penetrating urea. The intracellular Ca2+-ATPase inhibitor thapsigargin suppressed both the spontaneously occurring transients and those initiated by volume expansion. Touch stimuli induced [Ca2+]i transients, which rapidly propagated to cells within the same aggregate or lacking contact. CONCLUSION: The observations support the idea that beta-cells both receive and regenerate extracellular signals triggering [Ca2+]i transients. Touch stimulation is a useful tool for investigating the propagation of [Ca2+]i signals between pancreatic beta-cells lacking physical contact.