缩胆囊素和促胰液素对豚鼠离体胃平滑肌运动的影响

用８个肌槽同时记录豚鼠胃不同部位肌条的收缩活动,以观察八肽缩胆囊素（ＣＣＫ－８）和促胰液素的影响。结果表明：ＣＣＫ－８能（１）增高各部位纵行肌和环行肌的张力;（２）加快胃体纵行肌,胃窦纵行肌、环行肌和幽门环行肌的收缩频率;（３）增大胃窦环行肌收缩波平均振幅和（４）增加幽门环行肌收缩波运动指数,但减少胃体和胃窦纵行肌收缩波平均振幅。上述作用均不能被阿托品和消炎病所阻断。而促胰液素对各部位肌条的收缩活动没有明显的影响。     

In vitro response of rat gastrointestinal segments to cholecystokinin and bombesin

In previous studies of the rat gastrointestinal (GI) tract, we have demonstrated specific binding of cholecystokinin (CCK) to the pylorus and of bombesin (BN) to the gastric fundus, gastric antrum, duodenum, and ileum. We now present the results of an investigation of the in vitro response of the same regions of the rat GI tract to CCK-8 (the active octapeptide of CCK) and BN. Sections of rat fundus, antrum, pylorus, duodenum, and ileum were suspended in a Tyrode buffer and attached to an isometric pressure transducer in a longitudinal orientation. Dose-response curves to CCK-8 and BN were generated for each tissue. CCK-8 consistently induced a change only in pylorus, while BN induced a response from fundus, antrum and duodenum. With the exception of the lack of ileal response to BN, the regions of the rat GI tract which biologically respond (i.e., contract or relax) to CCK-8 or BN were the same regions in which we have located BN and CCK-8 binding sites. This correlation supports the hypothesis that GI function is modified by specified hormone-receptor interactions with these peptides.     

Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.     

Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes

The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.     

Restraint stress augments postprandial gastric contractions but impairs antropyloric coordination in conscious rats

Central corticotropin-releasing factor (CRF) plays an important role in mediating restraint stress-induced delayed gastric emptying. However, it is unclear how restraint stress modulates gastric motility to delay gastric emptying. Inasmuch as solid gastric emptying is regulated via antropyloric coordination, we hypothesized that restraint stress impairs antropyloric coordination, resulting in delayed solid gastric emptying in conscious rats. Two strain gauge transducers were sutured onto the serosal surface of the antrum and pylorus, and postprandial gastric motility was monitored before, during, and after restraint stress. Antropyloric coordination, defined as a propagated single contraction from the antrum to the pylorus within 10 s, was followed by > or = 20 s of quiescence. Restraint stress enhanced postprandial gastric motility in the antrum and pylorus to 140 +/- 9% and 134 +/- 9% of basal, respectively (n = 6). The number of episodes of antropyloric coordination before restraint stress, 2.4 +/- 0.4/10 min, was significantly reduced to 0.6 +/- 0.3/10 min by restraint stress. Intracisternal injection of the CRF type 2 receptor antagonist astressin 2B (60 microg) or guanethidine partially restored restraint stress-induced impairment of antropyloric coordination (1.6 +/- 0.3/10 min, n = 6). The restraint stress-induced augmentation of antral and pyloric contractions was increased by astressin 2B and guanethidine but abolished by atropine, hexamethonium, and vagotomy. Restraint stress enhanced postprandial gastric motility via a vagal cholinergic pathway. Restraint stress-induced delay of solid gastric emptying is due to impairment of antropyloric coordination. Restraint stress-induced impairment of antropyloric coordination might be mediated via a central CRF pathway.     

Oxidative stress-induced calcium signalling in Aspergillus nidulans

The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

Effects of CCK-8 on the cytoplasmic free calcium concentration in isolated rat islet cells.

The C-terminal octapeptide of cholecystokinin (CCK-8) is known to stimulate insulin secretion. We examined its effects on the cytoplasmic free calcium concentration ([Ca2+]IC) in isolated rat pancreatic islet cells. At 8.3 mM glucose and 1.28 mM Ca2+, CCK-8 (100 nM) rapidly increased [Ca2+]IC to a short-lived peak, whereafter the [Ca2+]IC, within 1.5 minutes, fell to values below baseline. CCK-8 also rapidly increased the [Ca2+]IC at 3.3 mM glucose and in a calcium deficient medium. However, either at low glucose or in the absence of extracellular Ca2+, the post-peak [Ca2+]IC did not fall below baseline levels. The CCKA receptor antagonist, L-364,718 (20 nM), inhibited the effects of CCK-8 on [Ca2+]IC. The results suggest that CCK-8 in islet cells liberates calcium from intracellular stores by activating CCKA receptors.     

Ghrelin accelerates gastric emptying via early manifestation of antro-pyloric coordination in conscious rats

Ghrelin is known to enhance gastric motility and accelerate gastric emptying of liquid and solid food in rats. As solid gastric emptying is regulated by the coordinated motor pattern between the antrum and pylorus (antro-pyloric coordination), we studied the correlation between solid gastric emptying and antro-pyloric coordination in response to ghrelin. Rats were given 1.5 g of solid food after a 24-h fasting. Immediately after the ingestion, ghrelin (0.4-8.0 microg/kg) or saline was administered by intraperitoneal (i.p.) injection. Ninety minutes after the feeding, rats were euthanized and gastric content was removed to calculate gastric emptying. To evaluate the antro-pyloric coordination, strain gauge transducers were sutured on the antrum and pylorus. The incidence of postprandial antro-pyloric coordination was compared between ghrelin-and saline-injected rats. In saline-injected rats, gastric emptying was 58.3+/-3.7% (n=6). Ghrelin (4.0-8.0 microg/kg), accelerated gastric emptying. Maximum effect was obtained by ghrelin (4.0 microg/kg), which significantly accelerated gastric emptying to 77.4+/-3.7% (n=6, p<0.05). The number of antro-pyloric coordination 20-40 min after feeding was significantly increased in ghrelin-injected rats, compared to that of saline-injected rats (n=4, p<0.05). It is suggested that enhanced antro-pyloric coordination play an important role in accelerated solid gastric emptying induced by ghrelin.     

Immunostimulatory flagellin from Burkholderia pseudomallei effects on an increase in the intracellular calcium concentration and up-regulation of TNF-alpha by mononuclear cells

Using flow cytometry analysis, the flagellin of Burkholderia pseudomallei acts as a signalling inducer, and evokes an increase in the intracellular calcium ion concentration ([Ca(2+)]i) in human peripheral blood mononuclear cells (PBMC). The cells with increased [Ca(2+)]i segregate into the live monocyte gate and not into the live lymphocyte gates. The stimulated [Ca(2+)]i increase can be neutralized with anti-flagellin antibodies. In the absence of [Ca(2+)], [Ca(2+)]i was increased rapidly in flagellin-treated cells compared to non-flagellin-treated cells only after the addition of 1 mM CaCl(2). Selective calcium antagonists were used to effectively block the [Ca(2+)]i signal, revealing that this signal was decreased by the addition of L-type calcium channel blockers (diltiazem, nifedipine and verapamil) and La(2+) but was not changed by the addition of a T-type calcium channel blocker (flunarizine). It seemed that flagellin facilitates [Ca(2+)]i influx via a La(2+) sensitive L-type cellular membrane channel. Furthermore, flagellin also acts as a TNF-alpha inducer in a time- and concentration-dependent manner when adhered mononuclear cells are treated with flagellin. This ability to induce TNF-alpha production was affected by the presence of [Ca(2+)] in the culture medium. It suggested that B. pseudomallei flagellin is an immuno-stimulatory molecule, causing an increase in [Ca(2+)]i and an up-regulation of TNF-alpha, which may play an important role in the inflammation process.     

Cholecystokinin-8 induces intracellular calcium signaling in cultured myenteric neurons from neonatal guinea pigs

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca(2+)](i)). CCK-8 (10(-10)-10(-6)M) evoked concentration-dependent increases in percentage of neurons responding (8-52%) and delta[Ca(2+)](i) (76-169 nM). Gastrin (1 microM) also induced an increase in [Ca(2+)](i) in 29+/-6% of neurons (delta[Ca(2+)](i): 71+/-3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca(2+)](i) response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca(2+)](i) increments, as did the addition of the calcium channel inhibitors nickel (1mM) and lanthanum (5mM). Nifedipine (1-50 microM) dose-dependently attenuated CCK-caused [Ca(2+)](i) responses. CCK evokes [Ca(2+)](i) signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.     

Calcium activation of mougeotia potassium channels

Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation.     

Effects of cholecystokinin on appetite and pyloric motility during physiological hyperglycemia

Recent studies suggest that the interaction between small intestinal nutrient stimulation and the blood glucose concentration is important in the regulation of gastric motility and appetite. The purpose of this study was to determine whether the effects of cholecystokinin octapeptide (CCK-8) on antropyloric motility and appetite are influenced by changes in the blood glucose concentration within the normal postprandial range. Seven healthy volunteers were studied on 4 separate days. A catheter incorporating a sleeve sensor was positioned across the pylorus, and the blood glucose was stabilized at either 4 mmol/l (2 days) or 8 mmol/l (2 days). After the desired blood glucose had been maintained for 90 min, an intravenous infusion of either CCK-8 (2 ng. kg(-1). min(-1)) or saline (control) was given for 60 min. Thirty minutes after the infusion began, the catheter was removed and subjects drank 400 ml of water with guar gum before being offered a buffet meal. The amount of food consumed (kcal) was quantified. The order of the studies was randomized and single-blinded. There were fewer antral waves at a blood glucose of 8 than at 4 mmol/l during the 90-min period before the infusions (P<0.05) and during the first 30 min of CCK-8 or saline infusion (P = 0.07). CCK-8 suppressed antral waves (P<0.05), stimulated isolated pyloric pressure waves (IPPWs) (P<0.01), and increased basal pyloric pressure (P<0.005) compared with control. During administration of CCK-8, basal pyloric pressure (P<0.01), but not the number of IPPWs, was greater at a blood glucose of 8 mmol/l than at 4 mmol/l. CCK-8 suppressed the energy intake at the buffet meal (P<0.01), with no significant difference between the two blood glucose concentrations. We conclude that the acute effect of exogenous CCK-8 on basal pyloric pressure, but not appetite, is modulated by physiological changes in the blood glucose concentration.     

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.     

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated for 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By contrast, glutathione had no effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.     

Normalization of cytoplasmic calcium response in pancreatic beta-cells of spontaneously diabetic GK rat by the treatment with T-1095, a specific inhibitor of renal Na+-glucose co-transporters.

Chronic hyperglycemia is known to lead to a progressively further impaired insulin response and to hasten the development of complications in patients with type 2 diabetes, a notion referred as glucose toxicity. T-1095, a derivative of phlorizin, is a newly developed oral hypoglycemic agent that acts as a specific inhibitor of renal Na(+)-glucose co-transporters, reducing circulating blood glucose levels by promoting glucose excretion into urine. The effects of glycemic improvement by T-1095 on secretory function and cytoplasmic calcium response in pancreatic beta-cells were investigated using spontaneously diabetic GK rats. After four weeks of treatment with T-1095 (age 4 to 8 week rats), serum glucose and HbA1c levels were significantly improved (serum glucose level, GK vs. GK T-1095, 277.3 +/- 11.8 vs. 204.7 +/- 6.4 mg/dl; HbA1c level, GK vs. GK T-1095, 6.2 +/- 0.2 vs. 4.8 +/- 0.1 %). Insulin secretion induced by 16.7 mM glucose was also significantly increased in the T-1095-treated group compared to the untreated group. The [Ca(2+)]i response induced by 16.7 mM glucose in GK beta-cells was characterized by the loss of the steep first peak of [Ca(2+)]i elevation, and the lost first peak of [Ca(2+)]i reappeared in T-1095-treated beta-cells in 32 of 34 observations. In T-1095-treated beta-cells, the time lag to peak [Ca(2+)]i levels in the 16.7 mM glucose stimulation was significantly reduced (259.1 +/- 15.3 sec, p < 0.01) compared to untreated GK rats (524.7 +/- 52.9 sec). Thus, improvement of hyperglycemia by T-1095 ameliorates beta-cell function by relieving [Ca(2+)]i response.     

Preconditioning limits mitochondrial Ca(2+) during ischemia in rat hearts: role of K(ATP) channels

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca(2+)]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca(2+)]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca(2+)]m during ischemia-reperfusion and that this effect was mediated by opening mitochondrial K(ATP) channels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial K(ATP) channel opening (diazoxide, 100 microM) and blockade [5-hydroxydecanoic acid (5-HD), 100 microM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca(2+)]i and [Ca(2+)]m were measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca(2+)]m during ischemia-reperfusion. In addition, PC lowered [Ca(2+)]i during reperfusion, whereas diazoxide paradoxically resulted in increased [Ca(2+)]i during reperfusion. There was an inverse linear relationship between [Ca(2+)]m and developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca(2+)]i, an effect mediated by opening mitochondrial K(ATP) channels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.