Stimulatory effect of ghrelin on isolated porcine somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca(2+) concentration - [Ca(2+)](i) - in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca(2+)](i) in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys(3))-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca(2+)](i) evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca(2+)](i). The stimulatory effect of ghrelin on somatotropes was greatly attenuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na(+) solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca(2+)](i) transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca(2+)](i) similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca(2+)](i). Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca(2+)](i). Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.     

Subpopulations of somatotropes with differing intracellular calcium concentration responses to secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca2+]i). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Somatotropes were identified and distinguished from other adenohypophyseal cells, by their unique ability to respond to GH-releasing hormone with increased [Ca2+]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca2+]i (222 +/- 16 nM) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca2+]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca2+]i in only 21% of somatotropes. Perfusion with gonadotropin-releasing hormone elevated [Ca2+]i, but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogues.     

Effects of verapamil, dibutyryl cyclic AMP, and extracellular calcium on intracellular free calcium concentrations in myocytes isolated from rat ventricles

The concentration of free calcium in the myoplasm of myocytes isolated from rat ventricles was measured using quin2. Incubation of myocytes with the acetoxymethylester of quin2 (50 mumol.litre-1) for 45 min was required to give effective loading with quin2. At 1 mmol.litre-1 extracellular calcium free calcium was 210(17) (n = 5) nmol.litre-1. Depolarisation of myocytes by addition of 40 mmol.litre-1 potassium caused a threefold increase in free calcium. Increases in extracellular calcium from 0.25 to 2.0 mmol.litre-1 caused a twofold increase in free calcium in polarised (resting) myocytes and a 2.5-fold increase in cells depolarised with potassium. The ability of verapamil to inhibit the electrically stimulated contraction of myocytes was dependent on the stimulation voltage. Half-maximal inhibition was given by 0.7 and 2 mumol.litre-1 verapamil at 50 and 100 V stimulation respectively. High concentrations of verapamil completely inhibited potassium induced increases in the fluorescence of quin2 loaded cells. Half-maximal inhibition was observed at 0.5 mumol.litre-1 verapamil. The calcium agonist CGP 28392 enhanced potassium induced fluorescence of quin2 loaded cells. Dibutyryl cyclic adenosine monophosphate and adrenaline increased the proportion of rod shaped cells that contracted in response to stimulation by low, but not high, voltage. Dibutyryl cyclic AMP caused a threefold increase in the potassium induced increase in free calcium. It is concluded that verapamil decreases and that calcium agonists, dibutyryl cyclic AMP, and increases in extracellular calcium increase free calcium, as monitored by intracellular quin2.     

Effects of metabolic blockade on intracellular calcium concentration in isolated ferret ventricular muscle

Tension and intracellular free calcium concentration [( Ca2+]i) were measured in isolated ferret papillary muscles. When both anaerobic glycolysis and oxidative phosphorylation were prevented (metabolic blockade), there was a rapid decline of both developed tension and systolic [Ca2+]i signals. Subsequently, resting tension increased, and after a further delay, resting [Ca2+]i also rose. When oxidative metabolism was restarted after a period of metabolic blockade that was sufficient to elevate both resting tension and [Ca2+]i, a variable recovery of mechanical function occurred. In preparations that showed recovery, resting tension declined toward control level, and there was considerable recovery of developed tension. [Ca2+]i initially fell, but it then rose to a level similar to that at the end of the preceding period of metabolic blockade and exhibited large variations in amplitude with frequency components in the range 0.2-1 Hz. This elevated [Ca2+]i gradually declined. Arrhythmias were often present during this recovery period and appeared to be triggered by the spontaneous increases in [Ca2+]i. In preparations that failed to recover, resting tension remained elevated or increased, and developed tension showed little recovery. Such preparations showed larger rises in [Ca2+]i both during and after metabolic blockade, and [Ca2+]i continued to rise when oxidative metabolism was restarted. In experiments in which Na-Ca exchange was inhibited (by replacement of sodium by lithium or by the application of nickel), the rise of [Ca2+]i when oxidative metabolism was restarted was reduced, but recovery of mechanical function was improved. The correlation between elevated [Ca2+]i on reactivation of oxidative metabolism and failure of recovery of mechanical function suggests that elevated [Ca2+]i has a direct role in preventing the recovery of mechanical function.     

Mechanism of action of the growth hormone secretagogue,L-692,585, on isolated porcine somatotropes

The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 microM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca(2+)](i)) of 53+/-1 nM (mean+/-S.E.M.) (P < 0.01) and 68+/-2 nM (P < 0.01) respectively. The L-585 response was characterized by an initial increase in [Ca(2+)](i) followed by a decline to a plateau level above the baseline. Concurrent calcium imaging with RHPA indicated that the L-585-evoked increase in [Ca(2+)](i) coincided with GH release. L-585 significantly increased the percentage of plaque-forming cells (24+/-3 vs 40+/-6%; P < 0.05) and mean area of plaques (1892+/-177 vs 3641+/-189 micro m(2); P < 0.01) indicating increased GH release. Substance P (SP) analogue ([d -Arg(1),d -Phe(5),d -Trp(7,11)]-SP) blocked, and the hGHRH receptor antagonist ((Phenylac-Tyr(1),d -Arg(2), p-chloro-Phe(6), Homoarg(9), Tyr (Me)(10), Abu(15), Nle(27),d -Arg(28), Homoarg(29))-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRH-induced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca(2+)](i) transient, indicating the involvement of adenylate cyclase-cAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca(2+)](i) whereas calcium influx during the second phase is a consequence of somatotrope depolarization.     

Effects of low temperature on intracellular ionic concentrations and transmembrane potential in isolated rabbit hearts

The effects of lowering the temperature from 33° to 23°C on the electrical activity and intracellular Na and K concentrations of isolated rabbit hearts were studied. The preparations were at a steady state for experimental periods of up to 80 min insofar as the extracellular space and the ionic content is concerned. Low temperature produced a decrease in intracellular sodium in both ventricles and an increase in intracellular potassium in the left ventricle. These ionic shifts did not alter the resting potential. The action potential amplitude and the overshoot were higher at 23°C than at 33°C. The maximum depolarization rate decreased and the action potential duration alternated regularly at 23°C. The decrease in $\text{V}\text{?}{}_{\mathrm{<mtext>max</mtext>}}$ is explained by a shift of the h_∞ vs V curve towards more negative potentials. It is postulated that the cyclic changes in duration are due to the temperature and frequency dependence of the outward currents I_K2 and I_x1 respectively.     

Effects of electrical stimulation and an intracellular calcium chelator on calcium movement in suspensions of isolated myocardial muscle cells

The procedure of Haworth RA, Hunter DR and Berkoff HA (J Mol Cell Cardiol, 1980; 12:715-23) for the isolation of myocardial muscle cells from rat hearts has been modified by the addition of a step which involves centrifugation of the cells through a Percoll gradient. This increased the proportion of rod-shaped cells from 47 +/- 2.2 to 80 +/- 1.3% (mean +/- SEM, n = 7). In the absence of electrical stimulation but in the presence of 1.3 mmol . litre-1 Ca2+ less than 2% of the cells beat spontaneously. This number was not increased by addition of the Ca2+-selective ionophore A23187. A method in which isolated myocytes suspended in a cyclindrical incubation chamber are stimulated to beat by electrical impulses is described. At 1.3 mmol . litre-1 extracellular Ca2+, electrical stimulation increased by 30% the amount of 45Ca2+ exchanged in the period 0.25 to 3 min following addition of 45Ca2+. For myocytes subjected to electrical stimulation, the amount of 45Ca2+ exchanged increased as the concentration of extracellular Ca2+ increased. At 0.5 mmol . litre-1 extracellular Ca2+ verapamil reduced the amount of 45Ca2+ exchanged by 15% while La3+ reduced the amount of 45Ca2+ exchanged by 80%. Incubation of myocytes with the acetoxymethyl ester of the intracellular Ca2+ chelating agent bis (o-aminophenoxy)-ethane-N,N,N'N'-tetraacetic acid (BAPTA) for 45 min led to an inhibition of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone (LH) drives diverse intracellular calcium second messenger signals in isolated porcine ovarian thecal cells: preferential recruitment of intracellular Ca2+ oscillatory cells by higher concentrations of LH

The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 microg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85+/-5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1) [Ca2+]i oscillations with periodicities of 0.5 to 4.5 min(-1) (63+/-4.5%), 2) a [Ca2+]i spike followed by a sustained plateau (17+/-2.6%), 3) a [Ca2+]i spike only (5.8+/-2.6%); and 4) a [Ca2+]i plateau only (3.8+/-1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean [Ca2+]i interspike periodicity from 1.09+/-0.16 to 0.59+/-0.07 min(-1) (P < 0.05). Putative phospholipase C inhibition with U-73122 (10 microM) also abolished or frequency-damped LH-driven [Ca2+]i oscillations in 95+/-4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually [Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH's dose-responsive drive of [Ca2+]i oscillations in gonadal cells.     

Effects of high-intensity exercise on leptin and testosterone concentrations in well-trained males

Objective: A number of investigations have examined the effect of exercise on leptin concentrations, because leptin is associated with obesity, satiety, and reproductive function. High-intensity exercise is known to increase testosterone, an inhibitor of leptin. The objective of the study was to determine whether the leptin responses to a progressive, intermittent exercise protocol were related to serum testosterone concentrations. Most previous studies have examined leptin responses to low or moderately high exercise intensities. A second objective was to determine whether leptin responses were different than previous experiments using intermittent moderate and high-intensity exercise. Methods: Well-trained runners completed strenuous intermittent exercise consisting of treadmill running at 60, 75, 90, and 100% VO _2max and a subsequent resting control trial was also conducted. Results: There were significant increases in mean serum levels of leptin and testosterone with both quickly returning to baseline during recovery, but no relationship between the two hormones was found. After examining individual data for both hormones, it was discovered that subjects could be classified as leptin responders or nonresponders, whereas testosterone increased in all subjects. Responders had elevated serum leptin levels at baseline and exhibited increases after high-intensity exercise, whereas nonresponders did not show changes in leptin during exercise. Conclusions: Data suggest testosterone levels do not acutely affect leptin responses to exercise or 1-h of recovery. Moreover, varied leptin responses to intense exercise in comparable well-trained runners was observed and was associated with baseline leptin concentrations.     

Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells.

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70-140 nmol/l and maximal, carbachol-induced peaks reached 1.1 mumol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells.     

Effects of porcine follicular fluid on gonadotropin concentrations in rhesus monkeys

The administration of steroid-free charcoal-treated porcine follicular fluid to long term castrated female rhesus monkeys lowered basal serum concentrations of FSH and had almost no effect on serum LH. Treatment with porcine follicular fluid before the administration of exogenous LRH inhibited the release of FSH, but also affected the release of LH. This inhibition was especially striking on the suppression of the peak of release of both FSH or LH at 20 min. These findings suggest that an inhibin-like material present in follicular fluid could play an important role in the secretion of FSH and LH in rhesus monkeys.     

Membrane, intracellular, and plasma magnesium and calcium concentrations in preeclampsia

Changes in intracellular calcium and magnesium concentrations seem to be involved in the pathogenesis of preeclampsia, whereas the role of cell membranes has not been studied in detail yet. To investigate the changes in calcium and magnesium metabolism in normal pregnancy and preeclampsia, plasma, intracellular, and membrane calcium and magnesium concentrations were determined in a clinical study. Twenty-five control, 18 untreated healthy pregnant, and 16 nulliparas preeclamptic women were investigated. Plasma, cellular, and membrane (erythrocytes) calcium and magnesium contents were measured by atomic absorption spectroscopy. Plasma and intracellular magnesium concentrations were significantly lower in the healthy pregnant group and the preeclamptic group as compared to controls (P < .01). In erythrocyte membranes magnesium content was found significantly decreased in the preeclamptic women as compared to healthy subjects (P < .001). There was a significant decrease in the plasma calcium concentration in the preeclamptic group compared to controls or healthy pregnant women (P < .05). Membranous calcium content was significantly increased in the preeclamptic group versus controls or healthy pregnant women (P < .001) and an inverse correlation with membranous magnesium content was found (r = -0.79,P < .01). Lowered plasma, intracellular, and membrane magnesium concentrations in preeclampsia may contribute to the development in hypertension in pregnancy. In addition, a disturbed calcium homeostasis is observed in preeclampsia.     

The effects of age on platelet intracellular free calcium concentrations in normotensives and hypertensives.

We have investigated relationships between age, blood pressure and intracellular calcium concentration in platelets from normotensives and hypertensives. In normotensives, there were positive correlations between age and platelet intracellular calcium concentration (r = 0.76, P less than 0.001), age and mean arterial pressure (MAP; r = 0.55, P less than 0.01) and MAP and platelet intracellular calcium concentration (r = 0.45, P less than 0.01). Multiple regression analysis revealed that age was the primary determinant of platelet intracellular calcium concentration in normotensives. The effect of MAP on platelet intracellular calcium concentration when adjusted for age was not significant (P = 0.73). In hypertensives, there was no significant relationship between age and platelet intracellular calcium concentration (r = 0.15, P = 0.43), age and MAP (r = 0.17, P = 0.37) or MAP and platelet intracellular calcium concentration (r = -0.27, P = 0.15). Overall, platelet intracellular calcium concentration was significantly higher in hypertensives than in age-matched normotensives (P less than 0.05). Within the age groups examined, platelet intracellular calcium concentration was significantly higher only in younger hypertensives when compared with controls of a similar mean age (P less than 0.001). Thus, age, in addition to hypertension, is an important determinant of platelet intracellular calcium concentration.     

Changes in intracellular calcium during mechanical alternans in isolated ferret ventricular muscle

Alternans in heart is important as pulsus alternans in cardiac failure and electrophysiological alternans in myocardial ischemia. The explanation of this phenomenon is still unclear. We attempted to investigate the cellular mechanisms of alternans by measuring intracellular free calcium concentration [( Ca2+]i) with the photoprotein aequorin in isolated ferret papillary muscles. Tension and length were also recorded simultaneously. Transient mechanical alternans lasting five to 20 contractions could be reliably induced in this preparation by following a 30-second rest period with stimulation at a fast rate (2-4 Hz). Production of sustained mechanical alternans, which lasted longer than 20 contractions and could persist for several hundred contractions, required additional interventions, consisting of a lower temperature (25 degrees C), a lower external calcium concentration (1 mM), and a lower pH (6.91) than control conditions (0.33-0.5 Hz, 30 degrees C, 2 mM Ca2+, pH 7.36). Transient mechanical alternans was associated with transient in-phase alternation of aequorin light and, hence, [Ca2+]i. Sustained mechanical alternans was associated with sustained in-phase alternation of aequorin light as well as incomplete relaxation of tension. However, when muscles were switched from isometric to unloaded isotonic contraction, relaxation between stimuli was complete but contraction and the aequorin light signal continued to alternate. The addition of 10 mM caffeine or 10 microns ryanodine abolished transient and sustained mechanical alternans and also abolished the associated alternation of aequorin light. Commensurate with the action of ryanodine, which allows the sarcoplasmic reticulum to reaccumulate calcium to a limited extent after a period of rapid stimulation, sustained mechanical alternans sometimes reappeared in an attenuated form 30 to 50 contractions after the addition of ryanodine. These results demonstrate that incomplete muscle relaxation between beats need not be present for alternans to occur, and support the hypothesis that alternans is caused by intracellular calcium cycling involving the sarcoplasmic reticulum.     

Serum leptin concentrations in endometriosis

It has been recently reported that serum concentrations of the adipocyte-derived hormone leptin are increased in patients affected by endometriosis. On the basis of these findings, the present study was undertaken to evaluate whether the protein may be used as a new serum marker of the disease. A consecutive series of 67 reproductive-age women who underwent laparoscopy for benign gynecological pathologies were enrolled prospectively for the study. Serum leptin concentrations, as evaluated by a conventional RIA kit, were related to baseline clinical characteristics and surgical and histologic diagnosis. Endometriosis was documented in 42 women (stage I-II in 19 patients and stage III-IV in 23 patients). Twenty-five women of similar age and body mass index, who had no laparoscopic evidence of the disease, served as control group. Serum levels of leptin resulted similar between women without and with endometriosis at any stage (mean +/- SEM, 12.5 +/- 9.4 ng/ml and 12.1 +/- 8.0 ng/ml, respectively). No significant association with leptin concentrations was observed in regard to stage of the disease, number of endometriotic implants, presence/absence of an endometriotic cyst or peritoneal deep endometriosis, and presence/absence of specific symptoms. Therefore, our results do not support the possibility to employ leptin measurement as a diagnostic tool for endometriosis. Further studies are needed to elucidate the relationship between leptin and endometrial system and determine the potential contribution of the molecule in implantation and early pregnancy development.     

Changes in intracellular free calcium in isolated myometrial cells: role of extracellular and intracellular calcium and possible involvement of guanine nucleotide-sensitive proteins

Intracellular free calcium concentrations were measured directly in rat myometrial cells loaded with fura-2. The basal concentrations of calcium were 148 +/- 5.0 and 137 +/- 3.7 nM in the presence and absence of 1 mM extracellular calcium, respectively. Oxytocin, carbachol, and norepinephrine rapidly and transiently increased intracellular free calcium, with half-maximal effects at 0.19, 9.9, and 5.3 microM, respectively. The maximal effects of these agents were reduced by 57%, 32%, and 36%, respectively, when the extracellular calcium was replaced by 2 mM EGTA. Pretreatment with pertussis toxin partially (47-57%) inhibited the contractant-induced increase in intracellular free calcium in the presence of 1 mM extracellular CaCl2 and produced an even greater inhibition (76-98%) in the absence of extracellular calcium. Pretreatment with D600 (30 microM) or amiloride (50 microM) and reduction of extracellular sodium did not affect the oxytocin-induced calcium increase. However, adenosine and the A2-receptor agonist N-ethylcarboxamidoadenosine did attenuate the effect of oxytocin in a dose-dependent manner. These data represent the first direct evidence that oxytocin, carbachol, and norepinephrine increase the intracellular free calcium concentration in the rat myometrium. The data suggest that contractants mobilize calcium from both extracellular and intracellular sources, the latter involving a pertussis toxin-sensitive mechanism.     

玉郎伞多糖对Aβ25-35所致PC12细胞损伤细胞内钙离子浓度的影响

目的观察Aβ25-35诱导PC12细胞凋亡损伤模型胞内钙浓度的变化及YLSP的影响。方法采用Aβ25-35诱导PC12细胞凋亡损伤模型,石杉碱甲作为对照药,给予YLSP预处理后,加入Flu-3/AM,用流式细胞仪（FCM）测定各组细胞的平均荧光强度。结果各组PC12细胞内钙离子含量存在显著性差异（F=14.051,P〈0.01）,其中,Aβ25-35作用PC12细胞后,PC12细胞内钙离子含量显著升高（P〈0.01）,经YLSP处理后,PC12细胞内钙离子含量较模型组细胞显著降低（P〈0.01）,其中,YLSP高剂量组的PC12细胞内钙离子含量显著低于石杉碱甲组（P〈0.01）。结论 YLSP能对抗Aβ引起的细胞内钙离子浓度升高,防止钙超载的发生。     

Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes

OBJECTIVE: We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. METHODS: Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. RESULTS: PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. CONCLUSION: The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.