MIF-I suppresses deprivation-induced fluid consumption in rats

Rats were injected IP with a 0.1 mg/kg dose of MIF-I, naloxone, dynorphin, [D-Phe⁴]-Met-enkephalin, [D-Ala², F₅Phe⁴]-Met-enkephalin-NH₂, or the diluent vehicle, placed in their home cages for ten minutes, and then given ad lib access to either 20% sucrose, 10% sucrose, water, 0.01% quinine, or 0.02% quinine in a repeated measures design with solutions counter-balanced over five days. Fluid consumption was measured every hour for 4 hours. A mixed analysis of variance yielded significant results for all main effects and the peptides by fluid and hours by fluid interactions. For the 4-hr test period, naloxone and [D-Phe⁴]-Met-enkephalin produced reliable increases in consumption while MIF-I produced a reliable decrease. Differences were obtained only with sucrose solutions, and the results clearly suggest that peptides modulate fluid consumption at positive levels of incentive motivation. To reconcile the findings of increased consumption after naloxone with the many studies suggesting a decrease in such paradigms, 0.1, 1.0, and 10.0 mg/kg of naloxone and MIF-I were administered as before but to independent groups of rats and intake was measured every 30 min. These results replicate and extend the above findings by showing that during the first 30-min period, both naloxone and MIF-I suppressed intake in a dose-dependent fashion, with MIF-I being more effective at each dose. The 0.1 mg/kg naloxone group, however, increased consumption over time and achieved a total consumption greater than control animals but comparable to that observed in the first study. It appears that at very low doses naloxone increases consumption over time, but at more commonly tested higher doses it has a suppressant effect. The results support the concept that in many situations MIF-I can produce the same effects as naloxone.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

Dietary lipoic acid supplementation attenuates hypertension in Dahl salt sensitive rats

There is strong evidence that excess dietary salt (NaCl) is a major factor contributing to the development of hypertension. Salt sensitive humans and rats develop hypertension even on a normal salt diet. Salt sensitivity is associated with glucose intolerance and insulin resistance in both humans and animal models, including Dahl salt sensitive (DSS) rats. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes. These aldehydes bind sulfhydryl groups of membrane proteins, altering calcium channels, increasing cytosolic free calcium ([Ca²⁺]_i) and blood pressure. Treatment with lipoic acid, an endogenous sulfur-containing fatty acid, normalizes insulin resistance and lowers tissue aldehyde conjugates, cytosolic [Ca²⁺]_i, and blood pressure in spontaneously hypertensive rats (SHR). The objective of this study was to investigate the effects of a normal salt diet on tissue aldehyde conjugates, cytosolic [Ca²⁺]_i and blood pressure in DSS rats and to determine whether lipoic acid supplementation prevents the increase in blood pressure and biochemical changes. Starting at 7 weeks of age, DSS rats were divided into three groups of six animals each and treated for 6 weeks with diets as follows: DSS-low salt, 0.4% NaCl; DSS-normal salt, 0.7% NaCl, and; DSS-normal salt + lipoic acid, 0.7% NaCl + lipoic acid 500 mg/kg feed. At completion, animals in the normal salt group had elevated systolic blood pressure, cytosolic [Ca²⁺]_i and tissue aldehyde conjugates as compared to the low salt group. They also showed smooth muscle cell hyperplasia in small arteries and arterioles of the kidney. Dietary lipoic acid supplementation attenuated the increase in systolic blood pressure and associated biochemical and histopathological changes.     

K homeostasis and central pattern generation in the metathoracic ganglion of the locust

Stress-induced arrest of ventilatory motor pattern generation is tightly correlated with an abrupt increase in extracellular potassium concentration ([K⁺]_o) within the metathoracic neuropil of the locust, Locusta migratoria. Na⁺/K⁺-ATPase inhibition with ouabain elicits repetitive surges of [K⁺]_o that coincide with arrest and recovery of motor activity. Here we show that ouabain induces repetitive [K⁺]_o events in a concentration-dependent manner. 10⁻⁵ M, 10⁻⁴ M, and 10⁻³ M ouabain was bath-applied in semi-intact locust preparations. 10⁻⁴ M and 10⁻³ M ouabain reliably induced repetitive [K⁺]_o events whereas 10⁻⁵ M ouabain had no significant effect. In comparison to 10⁻⁴ M ouabain, 10⁻³ M ouabain increased the number and hastened the time to onset of repetitive [K⁺]_o waves, prolonged [K⁺]_o event duration, increased resting [K⁺]_o, and diminished the absolute value of [K⁺]_o waves. Recovery of motor patterning following [K⁺]_o events was less likely in 10⁻³ M ouabain. In addition, we show that K⁺ channel inhibition using TEA suppressed the onset and decreased the amplitude of ouabain-induced repetitive [K⁺]_o waves. Our results demonstrate that ventilatory circuit function in the locust CNS is dependent on the balance between mechanisms of [K⁺] accumulation and [K⁺] clearance. We suggest that with an imbalance in favour of accumulation the system tends towards a bistable state with transitions mediated by positive feedback involving voltage-dependent K⁺ channels.     

Discovery of pyrrolo[2,3-d]pyrimidin-4-ones as corticotropin-releasing factor 1 receptor antagonists with a carbonyl-based hydrogen bonding acceptor

A new class of pyrrolo[2,3-d]pyrimidin-4-one corticotropin-releasing factor 1 (CRF₁) receptor antagonists has been designed and synthesized. In general, reported CRF₁ receptor antagonists possess a sp²-nitrogen atom as hydrogen bonding acceptor (HBA) on their core scaffolds. We proposed to use a carbonyl group of pyrrolo[2,3-d]pyrimidin-4-one derivatives as a replacement for the sp²-nitrogen atom as HBA in classical CRF₁ receptor antagonists. As a result, several pyrrolo[2,3-d]pyrimidin-4-one derivatives showed CRF₁ receptor binding affinity with IC₅₀ values in the submicromolar range. Ex vivo ¹²⁵I-sauvagine binding studies showed that 2-(dipropylamino)-3,7-dimethyl-5-(2,4,6-trimethylphenyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (16b) (30 mg/kg, po) was able to penetrate into the brain and inhibit radioligand binding to CRF₁ receptors (frontal cortex, olfactory bulb, and pituitary) in mice. We identified pyrrolo[2,3-d]pyrimidin-4-one derivatives as the first CRF₁ antagonists with a carbonyl-based HBA.     

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca²⁺ oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca²⁺] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca²⁺ oscillations in the cytosol that are transduced into mitochondrial Ca²⁺ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]_mito and [ATP]_cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca²⁺]_cyto and [ATP]_cyto during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.     

Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells

Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca²⁺]_c, which was completely attenuated by removing Ca²⁺ from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca²⁺]_c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca²⁺]_c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.     

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10⁻⁶ M LTD₄, LTB₄ and FMLP with an increase in both InsP formation and [Ca²⁺]_i. FMLP caused a greater rise in InsPs than either LTD₄ or LTB₄, which were equivalent. LTD₄, however, caused a greater increase in [Ca²⁺]_i than LTB₄ (4-fold) or FMLP. The FMLP [Ca²⁺]_i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10⁻⁷ M for 3 min). In contrast, the LTD₄ [Ca²⁺]_i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca²⁺]_i and InsP responses to LTD₄ by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD₄ evoked increases in [Ca²⁺]_i.     

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .