TNF-alpha augments the expression of RhoA in the rat bronchus.

While nonspecific airway hyperresponsiveness (AHR) is a central feature of allergic bronchial asthma, the mechanism underlying the development of AHR is not clearly understood. We have previously demonstrated in vitro hyperresponsiveness of bronchial smooth muscle to acetylcholine (ACh) in rats that were actively sensitized and repeatedly challenged with aerosolized antigen. It has also been demonstrated that the ACh-induced, RhoA-mediated Ca(2+) sensitization is markedly augmented concomitantly with an increased expression and activation of RhoA protein in the bronchial smooth muscle of the antigen-treated rats. In the present study, we have investigated whether TNF-alpha, a proinflammatory cytokine which is involved in bronchial asthma, causes upregulation of RhoA mRNA and protein in the rat bronchus. Treatment of rat bronchial smooth muscle preparations with TNF-alpha (300 ng/ml for 24 hr) significantly shifted the concentration-response curve to ACh upwards, but did not alter the response to high K(+), when compared to that of control tissues. Levels of RhoA mRNA and protein in the TNF-alpha-treated bronchus were significantly greater than those in the control group. In conclusion, it is suggested that the augmentation of the ACh-induced contractile response evoked by TNF-alpha might be mediated by an upregulation of RhoA in rat bronchial smooth muscle.     

Presynaptic inhibition of acetylcholine release

High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8-phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction.     

Does Ca2+ release by acetylcholine enhance the synthesis of inositol 1,4,5-trisphosphate in smooth muscle of the porcine coronary artery?

A possible role was investigated of the Ca2+ released by acetylcholine (ACh) in the ACh-induced synthesis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in smooth muscle of the porcine coronary artery. In Ca(2+)-free solution, 10 microM ACh transiently increased the cellular concentration of Ca2+ ([Ca2+]i) and Ins(1,4,5)P3. Divalent cation ionophores abolished the increase in [Ca2+]i but not the synthesis of Ins(1,4,5)P3 induced by subsequent application of 10 microM ACh in Ca(2+)-free solution, suggesting that the Ca2+ released by Ins(1,4,5)P3 following application of ACh does not act to accelerate the ACh-induced synthesis of Ins(1,4,5)P3 in smooth muscle of the porcine coronary artery.     

Cytosolic calcium levels in vascular smooth muscle

Increase in cytosolic Ca2+ level ([Ca2+] cyt) is prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+] cyt and muscle tension give direct information for the Ca2(+)-regulation of smooth muscle. A fluorescent Ca2+ indicator, fura-2, is used for this purpose. Comparison between [Ca2+] cyt and muscle tension in vascular smooth muscle indicates that, although high K+ and receptor-agonists such as norepinephrine and prostaglandin F2 alpha induce sustained contraction by the sustained increase in [Ca2+] cyt, greater contraction is produced by receptor-agonists than high K+ at a given [Ca2+]cyt. Phorbol ester show similar effects as receptor-agonists, and it potentiates a high K(+)-induced contraction with little effect on [Ca2+]cyt. These results suggest that the contraction of smooth muscle is due to the increase in [Ca2+]cyt. Furthermore, receptor-agonists stimulate phosphatidylinositol turnover and generates diacyl glycerol which activates protein kinase C and may consequently increase the Ca2+ sensitivity of contractile elements. The [Ca2+]cyt -dependent portion of these contractions is inhibited by Ca2+ channel blockers such as verapamil by the decrease in [Ca2+]cyt. By contrast, increase in cyclic AMP by isoproterenol and forskolin inhibits smooth muscle contraction by the decrease in [Ca2+]cyt also by the decrease in the Ca2+ sensitivity of contractile elements. Increase in the cyclic GMP level by sodium nitroprusside show effects quite similar to those of cyclic AMP. Thus, contractility of vascular smooth muscle seems to be regulated by [Ca2+]cyt and also by Ca2+ sensitivity of the contractile elements. Furthermore, at least part of the receptor-mediated changes may be due to activation of protein kinase C.     

Characterization of muscarinic receptors in rat bronchial smooth muscle in vitro

This study was undertaken to assess the important muscarinic receptor subtype in acetylcholine (ACh)-induced rat bronchial smooth muscle contraction. Ring smooth muscle strips of the left main bronchus were used. Isometrical contraction was measured in response to ACh in cumulative concentrations (10(-7)-10(-3) M) with and without preincubations with the muscarinic receptor antagonists, pirenzepine (an M1 antagonist), methoctramine (an M2 antagonist), and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; an M1/M3 antagonist). Preincubation with these antagonists resulted in concentration-dependent rightward shifts of the concentration-response curves to ACh. pA2 values (means+/-sem) were 8.80+/-0.10 for 4-DAMP, 7.03+/-0.06 for pirenzepine and 5.91+/-0.36 for methoctramine, indicating that the most important muscarinic receptor mediating ACh-induced contraction of rat bronchial smooth muscle is of the M3 type.     

Probable involvement of epsilon-isoform of protein kinase C in rat bronchial smooth muscle contraction induced by acetylcholine.

Although the role of protein kinase C (PKC) has been suggested in agonist-induced bronchial smooth muscle contraction, the PKC isoform(s) involved in this phenomenon is not clear now. In the present study, the effects of three PKC inhibitors, GF1092603X, G?6976 and rottlerin on acetylcholine (ACh)-induced bronchial smooth muscle contraction were examined to identify the PKC isoform(s) involved in the contraction. Bronchial smooth muscles were pretreated with each PKC inhibitor (10(-6) and 10(-5) M) 30 min before cumulative administration of ACh. In another series of experiments, the effects of PKC inhibitors on the maximal contraction induced by 10(-3) M ACh were determined: the inhibitors were cumulatively administered (10(-8)- 10(-5) M) after the ACh-induced contraction reached plateau. The ACh-induced bronchial smooth muscle contraction was significantly inhibited by GF109203X (inhibitor of PKC alpha, beta, gamma, delta and epsilon) but not by G?6976 (PKC delta, beta and gamma inhibitor) and rottlerin (PKC delta and theta inhibitor). Moreover, mRNA and protein of PKC epsilon were detected in rat bronchial smooth muscle. Taken together, PKC epsilon might be involved in the ACh-induced bronchial smooth muscle contraction in rats.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Calcium regulates L-Type Ca2+ channel expression in rat skeletal muscle cells

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.     

Potassium-depolarization-induced cytoplasmic [Ca2+] transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

The significance of voltage-activated Ca2+ currents in eliciting cytoplasmic Ca2+ transients was studied in pyramidal neurones isolated from the rat dorsal cochlear nucleus using combined enzyme treatment/mechanical trituration. Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) were evoked by K+-induced depolarizations (10-50 mM) and monitored by the Fura-2 fluorimetric technique. The acutely dissociated neurones had a resting [Ca2+]i of 17.2+/-0.5 nM. They possessed caffeine-sensitive Ca2+ stores which were empty at rest; these stores could be filled with Ca2+ entering from the extracellular space and were re-emptied quickly. The effects of various specific high-voltage-activated (HVA) Ca2+ channel antagonists (nifedipine, omega-agatoxin IVA and omega-conotoxin GVIA) on [Ca2+]i transients were tested. Analysis of the blocking effects of these agents on the [Ca2+]i, transients indicates that, in the pyramidal neurones of the dorsal cochlear nucleus, N-type Ca2+ channels are primarily responsible for producing the depolarization-induced increases in [Ca2+]i.     

Acetylcholine-induced asynchronous calcium waves in intact human bronchial muscle bundle

Calcium (Ca2+) is an important activator of the contractile machinery in airway smooth muscle (ASM). While agonist-induced Ca2+ signals are well characterized in animal ASM, little is known about what occurs in adult human ASM. In this study, we examined the Ca2+ signal elicited by acetylcholine (ACh) in smooth muscle cells of the intact human bronchial muscle strips obtained from fresh surgical specimens in relation to muscle contraction. We found that ACh induces repetitive Ca2+ waves that spread along the longitudinal axis of individual cells in the intact human bronchial smooth muscle strips. These Ca2+ waves display no apparent synchronization between neighboring cells, and their generation precedes force development. Comparison of the ACh concentration dependence of tissue contraction and selected parameters of the asynchronous Ca2+ waves (ACW) reveals that the graded force generation by ACh-stimulated human bronchial muscle strips is achieved by differential recruitment of cells to initiate Ca2+ waves and by enhancement of the frequency of ACW once the cells are recruited. Furthermore, pharmacologic characterization shows that the ACW are produced by repetitive cycles of SR Ca2+ release via ryanodine-sensitive channels followed by SR Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+ ATPase. Extracellular Ca2+ entry involving receptor-operated channels/store-operated channels, reverse-mode Na+/Ca2+ exchange, and to a lesser extent L-type voltage-gated Ca2+ channels is required to maintain the ACW. These findings for the first time demonstrate the occurrence and the role of ACW in excitation-contraction coupling in adult human ASM.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.     

Possible involvement of sodium pump in the relaxation of rat aorta induced by alpha-human atrial natriuretic polypeptide

In order to clarify whether the sodium handling of smooth muscle is associated with the relaxing action of alpha-human atrial natriuretic polypeptide (alpha-hANP), we examined the sodium pump-related effects of alpha-hANP on rat aortic smooth muscles. Application of Ca2+ (1.0 to 10.0 mM) to the muscle preincubated in Ca2+-free, and K+-free or 0.5 mM K+ medium for 60 min induced a contraction. Pretreatment with alpha-hANP (1 x 10(-8) M) decreased the contraction evoked in 0.5 mM [K+]o but not that in K+-free medium. After a contraction was elicited by norepinephrine in K+-free solution, an addition of KCl (1.4-5.4 mM) caused a transient relaxation in a concentration-dependent manner, presumably due to the activation of electrogenic Na pump. The alpha-hANP enhanced the relaxation, which was sensitive to ouabain, and the potentiation by alpha-hANP was inversely related to the concentration of K+ added. When alpha-hANP was applied to relax the muscle precontracted by norepinephrine in the varied concentration of external K+, alpha-hANP-induced relaxation was greater in 1.4 or 2.7 mM [K+]o than in 0 or 5.4 mM [K+]o. These results suggest that the vasodilating effect of alpha-hANP is at least partially mediated by the activation of electrogenic Na, K-pump and this effect is prominent when the Na, K-pump is partially suppressed.     

Elevated contractile responses to acetylcholine in organ cultured rabbit carotid artery

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum- free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Changes in calcium signalling in dorsal horn neurons in rats with streptozotocin-induced diabetes

Intracellular calcium signalling was studied in the dorsal horn from neurons of rats with streptozotocin-induced diabetes versus control animals. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2 acetoxymethyl ester-loaded dorsal horn neurons from acutely isolated spinal cord slices using a fluorescence technique. The recovery of depolarization-induced [Ca2+]i increase was delayed in diabetic neurons compared with normal animals. In normal neurons, [Ca2+]i after the end of KCl depolarization recovered to the basal level monoexponentially with a time constant of 8.0+/-0.5 s (n = 23), while diabetic neurons showed two exponentials in the [Ca2+]i recovery. The time constants of these exponentials were 7.2+/-0.5 and 23.0+/-0.6 s (n = 19), respectively. The amplitude of calcium release from caffeine-sensitive endoplasmic reticulum calcium stores became significantly smaller in diabetic neurons. The amplitudes of [Ca2+]i transients evoked by 30 mM caffeine were 268+/-29 nM (n = 13) and 31+/-9 nM (n = 17) in control and diabetic neurons, respectively. We conclude that streptozotocin-induced diabetes is associated with prominent changes in the mechanisms responsible for [Ca2+]i regulation, which presumably include a slowdown of Ca2+ elimination from the cytoplasm by the endoplasmic reticulum.     

Influences of ionic environments on ACh-induced secretory responses in isolated perfused pancreas of rats

The influence of extracellular Ca2+ concentration, [Ca2+]o, on the secretory response to acetylcholine (ACh) was analyzed in isolated perfused rat pancreas. The decrease of [Ca2+]o strongly diminished the amylase output and pancreatic juice flow in response to continuous stimulation with 5 X 10(-8) M ACh. A quantitative relation was found between the amount of amylase release by 5 X 10(-8) M ACh and the [Ca2+]o over a range of 0.1--2.5 mM. The partial replacement of NaCl with LiCl produced a diminution in both amylase output and pancreatic juice flow. A quantitative relation existed between the amount of ACh-induced amylase release and the [Na+]o over a range of 86--157 mM. The partial replacement of KCl with NaCl produced falls in both amylase output and pancreatic juice flow. Again, a quantitative relation existed between ACh-induced amylase release and [K+]o over a range of 1.0--5.6 mM. These results are compatible with the view that both the amylase output and the juice flow induced by 5 X 10(-8) M ACh are proportional to the amount of carrier-Ca complex and that the inward movement of the complex may be linked closely to the activation of Na pumps on the pancreatic acinar cell. A dose-response relation was found between the concentration of ACh and the amylase output. The relation was shifted to the left when 1 mU/ml cholecystokinin-pancreozymin (CCK-PZ) was added. A similar shift was observed when 1 mU/ml secretin was added. These results support the view that ACh, CCK-PZ, and secretin may activate the common cellular process in stimulus-secretion coupling, although these secretagogues may severally act on the different receptor sites.