Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Pancreatic beta-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients

Pancreatic β-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In this study, we compared changes of [Ca²⁺]_i in single β-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most β-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca²⁺]_i, a process counteracted by ryanodine. The β-cells from the obese mice were characterized by ample generation of [Ca²⁺]_i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca²⁺]_i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.     

Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human lutinizing-hormone receptor

We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca²⁺]_i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH β- or α-subunits, evoked specific [Ca²⁺]_i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca²⁺]_i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca²⁺]_i responses were observed in 12–14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca²⁺]_i signal amplitude), and eliciting a [Ca²⁺]_i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca²⁺]_i spike-and-lateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn²⁺ following the [Ca²⁺]_i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca²⁺]_i resoonse, and pertussis toxin (PTX) did not alter LH-stimulated [Ca²⁺]_i signaling. However, overnight preincubation with LH reduced the percentage of [Ca²⁺]_i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca²⁺]_i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca²⁺ signaling response by recruiting an increasing number of [Ca²⁺]_i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca²⁺]_i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca²⁺]_i signal.     

Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6

Summary We examined the relationship between cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and mitochondrial matrix Ca²⁺ concentration ([Ca²⁺]_m) in the pancreatic beta-cell line, MIN6. [Ca²⁺]_c was monitored in a single or a group (30 cells) of fura-2-loaded MIN6 cells, and [Ca²⁺]_m was measured in a group (1 × 10⁶ cells) of MIN6 cells stably transfected with aequorin targeted at the mitochondria. Exogenous ATP (0.25 mmol/l) produced a single transient increase in [Ca²⁺]_c whereas 22 mmol/l KCl produced a sustained plateau increase. ATP and KCl evoked transient increases in [Ca²⁺]_m but with distinct time courses of [Ca²⁺]_m decline: the [Ca²⁺]_m increase induced by ATP decreased more rapidly than that induced by KCl. Nitrendipine (3 μmol/l), a blocker of L-type Ca²⁺ channels, inhibited both [Ca²⁺]_c and [Ca²⁺]_m signals in response to KCl and tolbutamide, but not those to ATP. Peak levels of [Ca²⁺]_m increase (around 2 μmol/l) exceeded those of [Ca²⁺]_c increase (around 500 nmol/l). A rise in glucose concentration from 3 to 30 mmol/l induced oscillations of [Ca²⁺]_c that overlay the sustained increases in [Ca²⁺]_c in single cells. An oscillatory increase in [Ca²⁺]_m was similarly observed in response to glucose. Addition of 10 mmol/l 2-ketoisocaproic acid at 20 mmol/l glucose further increased the plateau level of [Ca²⁺]_c and the frequency of [Ca²⁺]_c oscillations, which were correlated with a further increase in [Ca²⁺]_m. In response to pulsatile exposure to KCl, [Ca²⁺]_c and [Ca²⁺]_m increased synchronously. These data suggest that an oscillatory increase in [Ca²⁺]_m in beta cells, the signal which is thought to be necessary for continuous stimulation of mitochondrial metabolism, is produced synchronously with the [Ca²⁺]_c oscillations. [Diabetologia (1998) 41: 279–286] Received: 24 July 1997 and in revised form: 27 October 1997     

Intracellular calcium ion responses to somatostatin in cells from human somatotroph adenomas

OBJECTIVE Various GH secretory responses to long-acting somatostatin (SRIH) analogues have been observed during the treatment of acromegalic patients. The effects of SRIH on intracellular Ca²⁺ homeostasis in human somatotroph adenoma cells has not been examined in detail, and the underlying mechanisms therefore remain to be determined. Using isolated cells from human somatotroph adenomas, we have investigated the SRIH-induced intracellular Ca²⁺ responses at a single-cell level with computerized real time intracellular calcium ion (Ca²⁺_i) imaging. PATIENTS Adenoma specimens were obtained from 4 male and 11 female acromegalic patients (mean age 56, range 26–72 years) undergoing transsphenoidal hypophysectomy. METHODS The identity of the biopsy material obtained was confirmed by immunocytochemistry for hGH and in situ hybridization histochemistry using a ³⁵S end-labelled hGH oligodeoxynucleotide probe and probes complementary to proopiomelanocortin and prolactin. Genomic DNA coding for somatostatin receptor (SSTR2) from each adenoma was PCR amplified and sequenced. Cells cultured from these adenoma were subject to computerized real time intracellular Ca²⁺_i imaging at a single cell level. RESULTS In cells from 11 of the 15 adenomas, SRIH produced a reversible, dose-independent reduction in [Ca²⁺]_i from the mean of 167±11 to 43±3 nM within 51±1.8s, and blocked the growth hormone releasing hormone (GRH)-induced increase in [Ca²⁺]_i as expected. In the same adenomas, withdrawal of SRIH after a 30 second exposure produced a small but significant increase in resting [Ca²⁺]_i. Pretreatment with pertussis toxin abolished the SRIH-induced inhibition of [Ca²⁺]_i and prevented the SRIH-induced inhibition of the effect of GRH on [Ca²⁺]_i. One of the remaining 4 adenomas was completely unresponsive to SRIH despite responding vigorously to other ligands and immunostaining strongly for GH. Surprisingly, cells from 3 adenomas showed a paradoxical increase in [Ca²⁺]_i in response to SRIH in some or, in one case, all of the cells examined. In all adenomas the sequence of SSTR2 corresponded to wild-type. CONCLUSIONS In the majority of cells derived from human somatotrophic adenomas, SRIH caused a reduction in baseline [Ca²⁺]_i and inhibition of GRH-induced [Ca²⁺]_i increase, as observed in somatotrophs of other species. In addition, SRIH was found either to induce a paradoxical increase in [Ca²⁺]_i or to have no effect on [Ca²⁺]_i in a small proportion of somatotroph adenomas examined. This finding corroborates the clinical observation that the response to SRIH analogues varies markedly between somatotroph adenoma patients. There was no evidence of SSTR2 mutations in any of the adenomas examined.     

Effects of new intravascular contrast agents on [Ca2+]i transients and contraction in cultured ventricular myocytes

Summary We examined the effects of four kinds of intravascular contrast agents (amidtrizoic acid, iohexol, iopamidol, and ioxaglic acid) on [Ca²⁺]_i transients (indo-1 fluorescence) and cell contraction (video motion analyzer), using cultured chick embryo ventricular myocytes. Exposure of ventricular myocytes to amidtrizoic acid (a conventional contrast agent) reduced the [Ca²⁺]_i transients and the sensitivity of the contractile elements to [Ca²⁺]_i. Ioxaglic acid (a low osmotic contrast agent) also reduced the [Ca²⁺]_i transients, but did not significantly change the sensitivity of the contractile elements to [Ca²⁺]_i. Neither iohexol nor iopamidol (nonionic contrast agents) reduced the [Ca²⁺]_i transients, but both significantly decreased the sensitivity of the contractile elements to [Ca²⁺]_i. A marked negative inotropic effect of amidtrizoic acid was caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by ioxaglic acid is possibly the result of calcium binding, but is not caused by hypertonicity. The negative inotropism produced by nonionic contrast agents (iohexol and iopamidol) was due to hypertonicity, but not due to alterations in the [Ca²⁺]_i transients.Exposure of ventricular myocytes to nonionic contrast agents (iohexol and iopamidol) slowed decay in the [Ca²⁺]_i transients with increased end-diastolic [Ca²⁺]_i. After washing out the nonionic contrast agents, these parameters returned to control levels. On the other hand, exposure to amidtrizoic acid decreased end-diastolic [Ca²⁺]_i without changing decay time in the [Ca²⁺]_i transients. After washing out amidtrizoic acid, there was a prolongation of half decay time in [Ca²⁺]_i transients with a significant increase in end-diastolic [Ca²⁺]_i and cell position. Diastolic dysfunction just after washout of amidtrizoic acid was possibly caused by an increase in [Na⁺]_i due to sodium influx during exposure to the contrast agent.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.     

Acute hypoxia activates store-operated Ca2+ entry and increases intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle cells

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.KEYWORDS : Calcium signaling, pulmonary venous smooth muscle (PVSM), store-operated Ca²⁺ entry (SOCE), intracellular Ca²⁺ concentration ([Ca²⁺]_i)     

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats

Summary We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca²⁺]_i) of cultured neonatal rat ventricular cells using micro-fluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 115% of control length), moderately so (to 115%–125% of control length), or extensively (to over 125% of the control length). “Quick stretches” (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca²⁺]_i in the early phase of stretch (30s-2 min), followed by a small but sustained increase during the late phase of stretch (5–10 min). The initial transient increase in [Ca²⁺]_i after the “quick stretch” was preserved in the presence of gallopamil (10⁻⁷M) or ryanodine (10⁻⁵ M), but was absent in Ca²⁺-free medium or in the presence of gadolinium (10⁻⁷M). The late or steady state [Ca²⁺]_i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca²⁺-free medium. A steady-state increase in [Ca²⁺]_i was also evoked by “slow stretch” in which cells were slowly pulled to the final length within 1–2min. As the presence of external Ca²⁺ was indispensable, increased trans-sarcolemmal Ca²⁺ influx appears to be involved in both initial and steady-state increases in [Ca²⁺]_i. The initial increase in [Ca²⁺]_i after the “quick stretch” can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.     

Nisoldipine blocks the increase of intracellular free calcium-ion concentration associated with elevated sodium–lithium countertransport activity in erythrocytes in patients with NIDDM

To understand the mechanism by which elevated sodium–lithium countertransport activity (SLC) associates with increased intracellular free calcium-ion concentration ([Ca²⁺]_i), we investigated the relationship between SLC and the effects of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and a Ca²⁺-channel blocker, nisoldipine, on [Ca²⁺]_i in erythrocytes from 48 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). There was a significant correlation between SLC and [Ca²⁺]_i. Nisoldipine in the incubation medium significantly decreased [Ca²⁺]_i, and there was a significant positive correlation between SLC and the degree of [Ca²⁺]_i decrease. When the [Ca²⁺]_o was elevated, [Ca²⁺]_i was significantly increased, but nisoldipine almost completely suppressed this increase of [Ca²⁺]_i. There was a significant positive correlation between SLC and the degree of the suppression. These data suggest that elevated SLC correlates with increased [Ca²⁺]_i, and that the increased [Ca²⁺]_i might be due to the increased Ca²⁺ influx through a dihydropyridine-sensitive Ca²⁺ pathway. © 1997 by John Wiley & Sons, Ltd.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Summary Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca²⁺ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca²⁺. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and constructed [Ca²⁺]_i-force relationships in normal (n=6) and myopathic human hearts (n=10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca²⁺]_i was 225±52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca²⁺]_i was significantly higher at 361±68 nM. Calcium-sensitizing agents that shift the [Ca²⁺]_i-force relationship toward lower [Ca²⁺]_i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca²⁺ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished actice force production.     

The Physiology of the Transplanted Small Bowel: An Overview with Insight into Graft Function

Nilsson J, Sjödin L, Gylfe E. Supramaximal inhibition of cholecystokinin-induced pancreatic amylase release involves desensitization to cytoplasmic Ca²⁺. Scand J Gastroenterol 1994;29:561-568.

Background: Cholecystokinin (CCK) is a major stimulant of pancreatic enzyme secretion. The dose-response relationship for CCK-induced secretion is bell-shaped, with a characteristic supramaximal inhibition. The mechanism for this inhibition has now been studied.

Methods: The kinetics of amylase release and the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]i) were recorded during stimulation of guinea-pig pancreatic acinar cells with different concentrations of cholecystokinin octapeptide (CCK-8) and the Ca²⁺ ionophore ionomycin. Results: Individual cells reacted with [Ca²⁺]_i oscillations at 10^?11 10^?10 M CCK-8 and with an initial peak followed by a sustained suprabasal level at 10 ^?9–10^?8 M of the agonist. The latter response was also seen in suspensions of acinar cells at all tested concentrations of CCK-8 and at 10^?6-10^?5 M of ionomycin. With increases of extracellular Ca²⁺ from 0.5 to 5.0 mM there was a rise of [Ca²⁺]_i during exposure to 10^?9-10^?8 M CCK-8 or 10^?5 M ionomycin but a paradoxical decrease at lower concentrations of CCK-8 or ionomycin. A dose-dependent increase of amylase release was seen at CCK-8 concentrations from 10^?11 to 10^?9M. At 10^?9-10^?8M CCK-8 secretion was characterized by an initial peak followed by a sustained phase. Whereas the initial peak of secretion remained unaffected by increasing CCK-8 from 10^?9 to 10^?8M, the sustained phase was inhibited (supramaximal inhibition). Increasing extracellular Ca²⁺ from 0.5 to 5.0 mM transiently enhanced secretion in response to 10^?9 M but lacked effect during supramaximal inhibition of secretion by 10^?8 M CCK-8.

Conclusions: Both initial and sustained CCK-8-stimulated amylase release increase with [Ca²⁺]_i. However, supramaximal inhibition of secretion was not due to a decrease of [Ca²⁺]_i but was characterized by desensitization to the stimulatory effect of [Ca²⁺]_i.     

Low Ambient [Cl<Superscript>−</Superscript>] Increases Ca<Superscript>2+</Superscript> Mobilization and Stimulates Nitric Oxide and Prostaglandin E<Subscript>2</Subscript> Production in Human Bronchial Epithelial Cells

Changes in ionic composition of airway surface fluid may modulate airway epithelial functions. We tested the hypothesis that fluctuations of ambient ionic composition could affect airway epithelial Ca²⁺ dynamics and Ca²⁺-dependent cellular functions, including NO release and PGE₂ production in vitro. The responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i) to changes in extracellular Cl⁻ and Na⁺ concentrations ([Cl⁻]_e, [Na⁺]_e) in the human bronchial epithelial cell line, 16HBE cells, were measured by the fura-2 method. The NO release to the medium after lowering [Cl⁻]_e was measured by an amperometric NO sensor. PGE₂ production was measured by radioimmunoassay. Changing to isotonic low [Cl⁻]_e solution by substitution with gluconate caused a sustained increase in [Ca²⁺]_i in a concentration-dependent manner, with the maximal [Ca²⁺]_i increase from the baseline level being 243 ± 110 nM with Cl-free solution. The effect was not altered by thapsigargin but abolished by EGTA and by Cl channel blockers, including diphenylamine-2-carboxylate, disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, and disodium cromoglycate. In contrast, the effect of reduction of [Na⁺]_e by substitution with N-methyl-D-glucamine⁺ on [Ca²⁺]_i was less than that of reduction of [Cl⁻]_e. The reduction of [Cl⁻]_e caused a concentration-dependent rise in NO contents in the medium and PGE₂ production. This release of NO was inhibited by EGTA but not by dexamethasone pretreatment. These results suggest that the decrease in ambient [Cl⁻] induces Ca²⁺ mobilization probably through Ca²⁺ influx, followed by the release of NO and PGE₂, thereby modulating various cellular functions.     

Effect of Nitric Oxide on Histamine-Induced Cytological Transformations in Parietal Cells in Isolated Human Gastric Glands

Previous studies have shown that nitric oxide (NO) inhibits histamine-induced gastric acid secretion in isolated human gastric glands. NO synthase has been found to be present in the human oxyntic mucosa and has been suggested to serve as a paracrine regulator of gastric acid secretion. Histamine stimulation of parietal cells induces cytoskeletal rearrangements, recruitment of H⁺/K⁺-ATPase-rich tubulovesicles to the apical membrane and expansion of intracellular canaliculi. The aim of the present study was thus to investigate (i) the effect of an NO donor on histamine-induced cytological transformations and (ii) the influence of increased [Ca²⁺]_i on NO-induced morphological changes in human parietal cells. Human gastric glands were isolated and subjected to the NO donor SNAP prior to histamine administration. [Ca²⁺]_i was increased by photolysis of the caged Ca²⁺ compound NP-EGTA. The distribution of F-actin, ezrin, and H⁺/K⁺-ATPase was assessed by confocal microscopy. Ultrastructural analysis was performed using transmission electron microscopy. SNAP did not influence the histamine-induced translocation of F-actin, ezrin, and H⁺/K⁺-ATPase but prevented an increase in the canalicular size. Elevation of [Ca²⁺]_i in resting cells was found to mimic histamine-induced intraparietal cell transformations; however, NO-induced parietal cell morphology was unaffected by a rise in [Ca²⁺]_i. These results indicate that NO inhibits secretion of fluid into the canalicular lumen without affecting membrane recruitment and that this effect is Ca²⁺-insensitive.     

Imaging of Ca2+ Transients in Endothelial Cells of Single Perfused Capillaries: Correlation of Peak [Ca2+]i with Sites of Macromolecule Leakage

Objective: To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca²⁺]_i, does not increase uniformly within microvessel endothelial cells. Methods: We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca²⁺]_i using a cooled CCD camera. Results: Control [Ca²⁺]_i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca²⁺]_i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca²⁺]_i (274 ± 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca²⁺]_i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increases in [Ca²⁺]_i were largest. Conclusions: These data indicate that variation in [Ca²⁺]_i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.     

Platelet Cytosolic free Calcium Before and After Antihypertensive Treatment in Perinephritis Hypertension of the Rabbit

Cytosolic free calcium concentration ([Ca²⁺]_i) in platelets has been reported to be elevated in human essential hypertension, to be positively correlated with blood pressure and to decrease with blood pressure reduction. However, some groups have been unable to confirm these findings in either humans or hypertensive rats. We have examined the relationship between platelet [Ca²⁺]_i and blood pressure in the perinephritis model of hypertension in the rabbit. In addition, the effects of both acute and chronic treatment with verapamil or prazosin were studied. Mean arterial pressure, heart rate and platelet [Ca²⁺]_i were measured before and after treatment. Platelet [Ca²⁺]_i was measured by the Quin 2 fluorescence technique. Platelet [Ca²⁺]_i was similar for the normotensive and hypertensive rabbits, and no correlation between platelet [Ca²⁺]_i and blood pressure was observed. None of the antihypertensive treatments produced a lowering of platelet [Ca²⁺]_i. Therefore we conclude that platelet [Ca²⁺]_i is unlikely to be a universally useful index of [Ca²⁺]_i in vascular smooth muscle of resistance vessels.