Influence of Anoxia and Dinitrophenol on Ca2+ Efflux and Phosphorylase a Activity in Rabbit Colon Smooth Muscle

Abstract: It was observed in earlier studies that when the phosphorylase a activity of rabbit colon smooth muscle was increased by anoxia or 2,4-dinitrophenol (DNP), the calcium content of the mitochondrial fraction decreased. Despite this, under basal conditions there was no significant increase in the Ca²⁺ content in the fraction consisting of microsomes and cytoplasm. In the present study it was therefore investigated whether mitochondrial Ca²⁺ released by anoxia and DNP is translocated from the smooth muscle cells into the extracellular fluid. The Ca²⁺ efflux from rabbit colon into a Ca²⁺-free Krebs-Ringer bicarbonate buffer was measured by using a Ca²⁺-selective electrode. Both anoxia and DNP (6.6 × 10^-5M) increased the Ca²⁺ efflux from the smooth muscle cells. The local anaesthetic D-mepivacaine, at a concentration of 1×10 ³M, reduced the increase in Ca²⁺ efflux and simultaneously enhanced the anoxic or DNP-induced rise in phosphorylase a activity. The replacement of external Na⁺ by choline was found to reduce the basal Ca²⁺ efflux and to moderately increase the phosphorylase a activity. On addition of DNP to the Na⁺-free and Ca²⁺-free buffer there was no significant change in the Ca²⁺ efflux, but the increase in phosphorylase a activity was greater than in the physiological buffer containing 137 mM Na⁺. These observations support the suggestion that anoxia and DNP, by releasing Ca²⁺ from the mitochondria, increase the phosphorylase a activity of smooth muscle.     

Evidence for an inherent,ATP-stimulated uptake of calcium into chromaffin granules

Summary Partially purified chromaffin granules (granular fraction), crude mitochondria (mitochondrial fraction) and a microsomal fraction were prepared from bovine adrenals by differential centrifugation and characterized by their catecholamine content, succinate dehydrogenase and glucose-6-phosphatase activity. During isotonic incubation with 0.1 mM ⁴⁵Ca²⁺ all fractions showed an uptake of ⁴⁵Ca²⁺, which was stimulated by ATP. In addition, after incubation the granular fraction was further fractionated by sucrose density gradient centrifugation (2.0–1.3 M sucrose; 170,000g·60 min). In some of these experiments the granular fraction was incubated simultaneously with ⁴⁵Ca²⁺ and [³H](-)noradrenaline to compare the uptake of both. The rate of uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction (characterized by the highest catecholamine content and the lowest succinate dehydrogenase activity) was doubled by ATP. The ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules was inhibited by N-ethylmaleimide (NEM) (0.1 mM), 2,4-dinitrophenol (DNP) (0.1 mM), azide (1 mM), carbonyl cyanide mchlorophenylhydrazone (CCCP) (20M), atractyloside (50 M), ruthenium red (40 M) and amobarbital (1 mM). This inhibition pattern was different from that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the microsomal fraction, but it was similar to that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction. However, the following differences are incompatible with the view that a mitochondrial contamination, with a highly active uptake, is responsible for the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules: a) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was insensitive to 1 mM amobarbital, whereas this agent inhibited the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules. b) Replacement of ATP by succinate stimulated the uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction only. c) The dependence of the ATP-stimulated uptake of ⁴⁵Ca²⁺ on the concentration of ATP (0.1–5 mM) was determined: while uptake into the 2.0 M sucrose fraction of chromaffin granules exhibited saturation kinetics, that into the mitochondrial fraction was linearly related to the concentration of ATP. Interestingly, uptake of ⁴⁵Ca²⁺ into those fractions of chromaffin granules that are known to be contaminated with mitochondria (1.6 M sucrose fraction) exhibited bot a saturable and a nonsaturable component. d) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was more sensitive to 0.1 mM DNP than the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules.The comparison of the ATP-stimulated uptake of ⁴⁵Ca²⁺ with that of [³H](-)noradrenaline into the 2.0 M sucrose fraction of chromaffin granules revealed that amobarbital, N,N-dicyclohexylcarbodiimide (DCCD), DNP and increasing concentrations of ATP had the same influence on both uptake processes. From our results we conclude that an inherent, ATP-stimulated uptake of ⁴⁵Ca²⁺ exists in chromaffin granules. The effects of agents on the uptake of ⁴⁵Ca²⁺ and/or [³H](-)noradrenaline into chromaffin granules are discussed with regard to the granular uptake mechanisms.This work was supported by the Deutsche ForschungsgemeinschaftSome results were reported at the 18th Spring Meeting 1977 of the Deutsche Pharmakologische Gesellschaft (Burger and Häusler 1977)     

Cyclic AMP as a Mediator of the Relaxing Action of Papaverine,Nitroglycerine, Diazoxide and Hydralazine in Intestinal and Vascular Smooth Muscle

Abstract: Papaverine and nitroglycerine relaxed rabbit colon muscle. The relaxation was preceded by an increase of the cyclic AMP content of the muscle. There was a correlation between the degree of relaxation and the increase of cyclic AMP. A reduction of ATP content was obtained with these drugs; this effect was not present in the Ca⁺⁺-poor preparation in spite of the fact that the cyclic AMP level was still increased. Papaverine but not nitroglycerine reduced the phosphodiesterase activity of the intact rabbit colon. In a homogenate of colon muscle a marked inhibition of phosphodiesterase activity was obtained. The phosphodiesterase activity from different cell fractions was also studied. Phosphodiesterase from the cytoplasmic fraction was activated by papaverine and nitroglycerine, while that of a Ca⁺⁺-binding microsomal fraction like that of mitochondria was inhibited. Diazoxide and hydralazine relaxed bovine mesenteric artery but not rabbit colon. The relaxation was associated and correlated with an increase in the cyclic AMP content. A reduction in the ATP content and an activation of phosphorylase a activity was also obtained; the ATP reduction was Ca⁺⁺-dependent. A weak inhibitory effect on phosphodiesterase activity was observed in hornogenate of the artery after diazoxide. Hydralazine activated phosphodiesterase in a homogenate of mesenteric artery in a low concentration but inhibited it in a high concentration. It is suggested that cyclic AMP mediates, at least partly, the relaxing action of these four drugs probably by stimulating a Ca⁺⁺-binding process in the smooth muscle cell.     

Effects of Ca2+ and Mg2+ ions on the interaction between 3H-histamine and the histamine receptor-rich microsomal fraction

The histamine receptor-rich microsomal fraction, which was obtained from the longitudinal muscle of the cat small intestine, was incubated with ³H-histamine in normal Locke-Ringer solution, Ca²⁺-free solution, Mg²⁺-free solution and Ca²⁺- and Mg²⁺-free solution for 5 or 10 min. The radioactivities of the histamine receptor-rich microsomal fraction in the experiments with normal Locke-Ringer solution, Ca²⁺-free solution and Mg²⁺-free solution were significantly greater than that in the experiment with Ca²⁺- and Mg²⁺-free solution. Ca²⁺ and Mg²⁺, however, were without any effects on the non-specific combination of ³H-histamine with the rat microsomal fraction and bovine serum albumin supposedly devoid of histamine receptors. These results indicate that the increase of radioactivity of the histamine receptor-rich microsomal fraction in the presence of Ca²⁺ and/or Mg²⁺ is due to the combination of histamine with its receptor in the microsomal fraction.     

CYP2E1 overexpression inhibits microsomal Ca-ATPase activity in HepG2 cells

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca²⁺-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca²⁺-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca²⁺-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca²⁺ or ATP showed that CYP2E1 overexpression produced a decrease in V_max but did not affect the K_m for either Ca²⁺ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2 mM N-acetylcysteine prevented the decrease in microsomal Ca²⁺-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca²⁺-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.     

Relationships between Ca2+ uptake by a microsomal fraction of guinea-pig taenia caecum and its relaxation

A microsomal fraction isolated from guinea-pig taenia caecum can accumulate calcium ions in the presence of ATP and MgCl₂. This calcium-accumulating ability depends on the ATP concentration in the reaction mixture, and its pH optimum is near 7·8. Procaine and quinidine inhibit Ca²⁺ accumulation by fraction and suppress the relaxation of this tissue induced by papaverine, isoprenaline and phenylephrine. These results suggest that Ca²⁺ accumulation by the microsomal fraction isolated from guinea-pig taenia caecum plays an important role during the relaxation of this smooth muscle. Cyclic adenosine 3′,5′-monophosphate (cAMP) does not accelerate Ca²⁺ accumulation by the microsomal fraction. The failure of cAMP to accelerate Ca²⁺ uptake by this fraction implies that some unknown steps or factors may exist between the intracellular increment of cAMP and the following relaxation of taenia caecum.     

Comparison of the Effect of DDE on the Ca Metabolism of the Eggshell Gland and its Subcellular Fractions of the Duck and the Domestic Fowl

Abstract: In a strain of ducks sensitive to the eggshell-thinning effect of p-p'-DDE, administration of 40 mg/kg of the compound in the food for 45 days reduced the eggshell index (EI) by 13% and the content of calcium in the fluid of the shell gland forming an eggshell by 36%, and raised the calcium content of the shell gland mucosa by 19%, compared with the control values. DDE inhibited the translocation (secretion) of calcium between the gland mucosa and the uterine cavity. The ATP-dependent binding of Ca²⁺ to the subcellular fractions of the gland mucosa was reduced in DDE-treated ducks. The Ca²⁺ binding to a microsomal subfraction (FI) rich in fragments of the plasma membrane was reduced by 16%, whereas that to a subfraction Fill which bound Ca²⁺ at a very high rate was reduced by 36%. The latter may contain calcium-secreting granules of the gland. In the mitochondrial fraction the Ca²⁺ binding was reduced by 35%. In the domestic fowl DDE did not lower EI or interfered with the translocation of calcium between the shell gland mucosa and uterine cavity. DDE administration increased the Ca²⁺ binding to FI by 26%; the binding to other subfractions was not changed significantly. DDE may interfere with the stimulus-secretion mechanism of the eggshell gland in ducks through its effect on Ca²⁺ binding.     

Effects of quinidine on calcium transport activities of the rabbit heart mitochondria and sarcotubular vesicles.

The effects of quinidine on the calcium-transporting properties of sarcotubular (heavy microsomal) and mitochondrial fractions of the rabbit heart were investigated and compared with those of procaine amide and lidocaine. High concentrations of quinidine (10^?4-10^?3 M) markedly decreased microsomal and mitochondrial calcium uptake; these inhibitory actions were observed at all concentrations of ATP but not when low concentrations of Mg²⁺ were used in the incubation medium. Calcium binding by mitochondrial fraction, unlike microsomal fraction, was also decreased by high concentrations of quinidine. Procainc amide (10^?3M) had no effect on microsomal or mitochondrial calcium uptake whereas lidocaine (10^?3 M) showed a stimulatory action only on the microsomal calcium uptake at initial intervals of incubation. The ATPase activities of both microsomal and mitochondrial fractions were inhibited by quinidine but not by procaine amide or lidocaine. Quinidine, unlike procaine amide and lidocaine. was also found to release about 15% of the calcium bound by microsomes and mitochondria. The results presented in this study suggest that quinidine impairs microsomal and mitochondrial calcium transport; however, other antiarrhythmic agents such as procaine amide and lidocaine, which in high concentrations also depress myocardial contractility, do not decrease calcium transport by heart subcellular particles.     

The influence of hexobarbitone on calcium ion movements in isolated heart muscle

In beating atria (frequency 180/min) sodium hexobarbitone (7·8 × 10^?4M) inhibited the rate of uptake of ⁴⁵Ca²⁺ from the medium without influencing the total calcium content of the tissues. Concomitantly, the so-called exchangeable calcium fraction was diminished although not at equilbrium. In resting left auricles the barbiturate did not affect ⁴⁵Ca⁺-uptake or the exchangeable calcium fraction. It seems likely that the barbiturate reduces membrane permeability towards Ca²⁺ during excitation without influencing the permeability at rest.     

Intracellular free calcium concentration in rat anterior pituitary cells as indicated by fura-2: effect of arginine-vasopressin

Summary Arginine-vasopressin (AVP) stimulates adrenocorticotropin and -endorphin release from corticotrophs of the anterior pituitary gland through mechanisms which are not initiated by an elevation of the cellular levels of adenosine-3,5-cyclic-monophosphate. In the present study the effect of AVP on the cytoplasmic concentrations of free calcium ions in rat anterior pituitary cells was examined. Cytosolic free Ca²⁺ concentrations were monitored directly using the new, intracellularly trapped fluorescent indicator fura-2. In cells incubated in medium containing 1.3 mmol/l Ca²⁺, AVP (100 nmol/l) caused an immediate elevation of the cytoplasmic Ca²⁺ concentration by about 50 nmol/l (P < 0.001). The intracellular Ca²⁺ levels remained elevated during the observation period of 2–3 min. This effect of AVP was blocked by a specific vasopressin antagonist. By contrast, the glucocorticoid dexamethasone did not affect the AVP-induced elevation of cytosolic Ca²⁺ concentration. When the cells were incubated in Ca²⁺-free medium (Ca²⁺ omitted, EGTA 2 mmol/l), the AVP-induced as well as the K⁺ depolarization-induced increase in free cytoplasmic Ca²⁺ were abolished, whereas the ionophore ionomycin evoked a rapid transient elevation of free Ca²⁺. The increase in cytoplasmic Ca²⁺ concentration induced by AVP was preserved in medium containing the calcium channel blockers Mg²⁺ (Mg²⁺ 31.2 mmol/l; Ca²⁺ 1.3 mmol/l) or nifedipine (1 mol/l). The potassium-evoked calcium signal was blocked by Mg²⁺ (31.2 mmol/l). We conclude that vasopressin induces a rapid rise in the cytoplasmic concentration of free calcium ions in corticotrophs. Vasopressin may mobilize calcium through mechanisms that neither are glucocorticoid-sensitive nor involve the influx of extracellular celcium through voltage-dependent calcium channels. The rise in Ca²⁺ concentration may be an early intracellular signal that links the cell-surface receptors for vasopressin to secretion of ACTH and -endorphin. Send offprint requests to W. Knepel     

Effect of Glyceryltrinitrate and 8–Br–cGMP on Tension and Phosphorylase a Activity in Vascular Smooth Muscle

Abstract: The aim of the present study was to examine the effect of glyceryltrinitrate (GTN) and 8–Br–cGMP on tension and cytosolic calcium concentration in pre–contracted bovine mesenteric arteries (BMA). The activity of glycogen phosphorylase a was used as a measure of the cytosolic calcium concentration. The activity of this enzyme is regulated by the cytosolic calcium concentration and/or cAMP. Since the cAMP level was not found to be affected by GTN–treatment, the use of phosphorylase a activity to monitor changes in the cytosolic calcium concentration can be justified. The vessels were contracted with phenylephrine (10 μM) or 100 mM K⁺–depolarization, which caused an increase in phosphorylase a activity. Addition of 1 μM GTN to the phenylephrine–contracted vessels resulted in a 3–4–fold rise in intracellular cGMP level, which was accompanied by a large decrease in tension and phosphorylase a activity. The K⁺–depolarized vessels, on the other hand, were largely resistant to the relaxant action of GTN, and there was only a slight reduction of the phosphorylase a activity. In phenylephrine–contracted vessels, made tolerant to GTN by incubation at elevated pH in the presence of GTN (0.44 mM), no changes in tension and phosphorylase a activity were seen after stimulation with a test dose of GTN (1 μM). The cGMP response was also markedly blunted in the tolerant vessels. Relaxation of phenylephrine–contracted BMA induced by 8–Br–cGMP (0.5 mM) was also accompanied by a reduction in phosphorylase a activity. The close association between the relaxation of BMA and the reduction in phosphorylase a activity suggests that GTN and 8–Br–cGMP exert their effects mainly by reducing the cytosolic free calcium concentration although a contributing effect on the contractile apparatus can not be excluded     

Modification of skeletal muscle sarcotubular Ca2+-stimulated adenosine triphosphatase activity by various agents.

Ca²⁺ -stimulated ATPase was isolated from the sarcotubular fraction of rabbit skeletal muscle, and the effects of various interventions on its activity were studied in the presence of different concentrations of calcium. Decreasing the pH of the incubation medium from 7.5 to 6.0 or the concentration of K⁺ from 100 to 40 mM decreased the enzyme activity. Different anions (I^?, SCN^?, Br^? and NO₃^?) and cations (Hg²⁺, La³⁺ Ni²⁺, Sr²⁺, Mn²⁺ and Co²⁺) were found to inhibit Ca²⁺-stimulated ATPase activity. Ca²⁺ antagonists (verapamil and D600) and β-adrenergic blocking agents (propranolol and practolol) also had inhibitory effects whereas cyclic AMP-protein kinase augmented the enzyme stimulation by calcium. These results implicate sarcotubular Ca²⁺ -ATPase as a possible site of action of various agents affecting the skeletal muscle function.     

Ca2+-uptake into noradrenaline-storing granules of bovine splenic nerves

Summary Noradrenaline-storing granules, a mitochondrial fraction and a microsomal fraction of bovine splenic nerve trunks were prepared by differential centrifugation. These particulate fractions were characterized by their noradrenaline content, succinate dehydrogenase and glucose-6-phosphatase activity. In the presence of ATP-Mg²⁺ all three fractions accumulated ⁴⁵Ca²⁺ during incubation with 0.1 mM ⁴⁵CaCl₂, buffered with potassium phosphate or glycylglycine (pH 7.5; 28°C). The accumulated ⁴⁵Ca²⁺ was not removable by EGTA, and the uptake was absent at 0°C or after destruction of the particles by sonication. The behaviour of the ⁴⁵Ca²⁺-uptake into all three fractions against varying ATP-concentrations, metabolic inhibitors (pentachlorophenol, desaspidine, 2,4-dinitrophenol, N-ethylmaleimide, p-chloromercuribenzoate, sodium azide, amobarbital) and drugs (phenoxybenzamine, verapamil, prenylamine, reserpine, bretylium, phentolamine) was studied. Under nearly all conditions there were differences between the ⁴⁵Ca²⁺-uptake into mitochondria and that into microsomes, which suggests two distinct uptake processes. The ⁴⁵Ca²⁺-uptake into the granule fraction behaved intermediate between the two other fractions under many conditions, but not under all. Therefore, it is not possible to explain the ⁴⁵Ca²⁺-uptake into the granule fraction as being due to contamination with mitochondria and microsomes; an inherent ATP-Mg²⁺-dependent ⁴⁵Ca²⁺-uptake into the nerve granules must be postulated, which is not directly coupled with the noradrenaline transport into these particles.A particulate fraction (14000–1000000 g), containing noradrenaline granules, was prepared from the vas deferens of the rat. Incubation with 5×10^–6 M (-)-noradrenaline and 0.1 mM ⁴⁵Ca²⁺ showed that the particles of this fraction take up noradrenaline and ⁴⁵Ca²⁺. The uptake of both was dependent on ATP-Mg²⁺. The ATP-Mg²⁺-dependent uptake of both noradrenaline and ⁴⁵Ca²⁺ was substantially reduced in the corresponding tissue fraction prepared from denervated vasa deferentia.This work was supported by the Deutsche ForschungsgemeinschaftPreliminary accounts were presented at the 12th spring meeting 1971 of the Deutsche Pharmakologische Gesellschaft (Burger and Philippu, 1971)     

Role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> content in Ca<Superscript>2+</Superscript> entry of bovine airway smooth muscle cells

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2⁺ channels with methoxyverapamil or by the sustained K⁺-induced depolarisation. The slow Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.     

Inhibition of microsomal and mitochondrial Ca2+-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners

 Recent studies from our laboratory indicate that polychlorinated biphenyl (PCB) congeners in vitro perturbed signal transduction mechanisms including cellular Ca²⁺-homeostasis and protein kinase C translocation. We have now investigated the structure-activity relationship (SAR) of three PCB mixtures, 24 PCB congeners and one dibenzofuran for their effects on microsomal and mitochondrial Ca²⁺-sequestration in rat cerebellum. Ca²⁺-sequestration by these intracellular organelles was determined using radioactive ⁴⁵CaCl₂. All three mixtures studied, Aroclor 1016, Aroclor 1254 and Aroclor 1260, were equally potent in inhibiting microsomal and mitochondrial Ca²⁺-sequestration with IC₅₀ values of 6–8 μM. 1,2,3,7,8-Pentachlorodibenzofuran had no effect on Ca²⁺-sequestration by these organelles. The SAR among the congeners revealed: (1) congeners with ortho-/meta- or ortho-, para-chlorine substitutions were the most potent in inhibiting microsomal and mitochondrial Ca²⁺-sequestration (IC₅₀=2.4–22.3 μM); (2) congeners with only para- but without ortho-substitutions were not effective in inhibiting Ca²⁺-sequestration by microsomes and mitochondria; (3) increased chlorination was not related to the effectiveness of these congeners. The present SAR studies indicate that the effects of most PCB congeners in vitro may be related to an interaction at specific sites having preference for low lateral substitution or lateral content (meta- or para) in the presence of ortho-substitution. Received: 10 March 1995 / Accepted: 4 July 1995     

Biphasic increase in cytosolic free calcium induced by bradykinin and histamine in cultured tracheal smooth muscle cells: is the sustained phase artifactual?

The effects of bradykinin (BK) and histamine on intracellular Ca²⁺ ([Ca²⁺]i) were studied in fura-2-loaded guinea-pig tracheal smooth muscle cells in culture. BK, at 10 nM, and histamine, at 100 M, induced a rise in [Ca²⁺]i which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca²⁺]_i is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histamine, was strongly inhibited by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, usually used to deplete certain intracellular Ca²⁺-stores. Ni²⁺ (4 mM) did not affect the transient phase but abolished the sustained phase when cells were stimulated by BK, further supporting the fact that the transient phase was only dependent on intracellular Ca²⁺ pools. The sustained phase was partially (for BK) and completely (for histamine) inhibited by 30 M Mn²⁺. This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn²⁺, indicating that the Mn²⁺ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter that extrudes fura-2) drastically inhibited the sustained phase by more than 77010 for BK and 88010 for histamine. Our results suggest that the effects of BK and histamine on airway smooth muscle cells are mediated via bradykinin B2 receptors and histamine H1 receptors, respectively whose activation allows the rapid transient rise in [Ca²⁺]i from thapsigargin-sensitive intracellular Ca²⁺ pools. The sustained phase is proposed to be drastically influenced by an acceleration of fura-2 extrusion during the increase of [Ca²⁺]_i via a probenecid-sensitive mechanism.Abbreviations BK bradykinin - [Ca²⁺]_i cytosolic free calcium - CICR Ca²⁺-induced Ca²⁺ release - DMEM Dulbecco's modified Eagle's medium - DTPA diethylenetriaminepentaacetic acid - EGTA ethylene glycol-bis(b-aminoethylether) N,N,N,N-tetraacetic acid - FCS fetal calf serum - HEPES 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethansulfonic acid - IP inositol phosphates - TG thapsigargin - TSMC tracheal smooth muscle cells Correspondence to: C. Bronner     

Different Mechanism of Relaxation Induced by Aporphine Alkaloids in Rat Uterus

Abstract— We have examined the uterine relaxant action of three aporphine molecules (S-glaucine, S-boldine and R-apomorphine) in two experimental conditions, with and without calcium in the bathing solution, and compared these effects with those obtained with the calcium antagonists verapamil and diltiazem. The present study shows that the alkaloids relax the uterine muscle but with different mechanisms of action. In Ca²⁺-containing solution all three alkaloids relaxed the uterus previously contracted by KCl or acetylcholine, but in Ca²⁺-free medium only R-apomorphine was able to relax oxytocin-induced contraction. The calcium antagonists, verapamil and diltiazem, relaxed KCl- or acetylcholine-induced contraction in Ca²⁺-containing solution, whereas they only relaxed oxytocin-induced contraction in Ca²⁺-free medium at much higher doses. These results suggest that glaucine and boldine behave as specific calcium entry blockers without affecting the contractile machinery or intracellular Ca²⁺ levels as apomorphine does. The absolute configuration (S-glaucine and S-boldine vs R-apomorphine) may account for this different action.     

The alpha-adrenergic control of rabbit liver glycogenolysis.

We have confirmed that the electrical stimulation of the splanchnic nerve in the rabbit causes glycogenolysis m a cyclic AMP-independent way as found by Shimazu and Amakawa [1]; glycogen phosphorylase (1,4-α-d-Glucan: orthophosphate α-glucosyltransferase, EC 2.4.1.1) was activated, but phosphorylase b kinase (ATP: phosphorylase b phosphotransferase, EC 2.7.1.38) was not. We could, however, not confirm the observation of a decrease in phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) activity. Pretreatment of the rabbits with the α-adrenergic blocking agent phentolamine prevented the splanchnic nerve stimulation from activating glycogen phosphorylase.The addition of norepinephrine (10^?7 M) to isolated rabbit hepatocytes activated glycogen phosphorylase without an activation of phosphorylase b kinase. At 10^?6 M, norepinephrine activated both enzymes. Phentolamine blocked the activation of glycogen phosphorylase by norepinephrine at 10^?7M but not at 10^?6M. Absence of Ca²⁺ from the incubation medium prevented norepinephrine (10^?7 M) from activating glycogen phosphorylase. The ionophore A 23187 also caused an activation of phosphorylase (but not of phosphorylase b kinase) provided that Ca²⁺ was present in the incubation medium. These data indicate that sympathetic nervous control of liver glycogenolysis is achieved, via α-adrenergic receptors, by an increased concentration of cytosolic Ca²⁺ ions which stimulate rather than activate phosphorylase b kinase. The neurotransmitter involved is most probably norepinephrine.     

Effects of chlorpromazine and imipramine on rat heart subcellular membranes

The effects of chlorpromazine and imipramine at concentrations ranging from 25 to 120 (μm on ATPase activities, as well as calcium binding and uptake abilities of the rat heart subcellular membranes, were studied in vitro. Chlorpromazine significantly decreased calcium binding. Mg²⁺ ATPase and Na⁺?K⁺ ATPase activities of the sarcolemmal fraction, whereas imipramine decreased calcium binding, Ca²⁺ ATPase and Mg²⁺ ATPase activities. Chlorpromazine also produced significant inhibition of the calcium binding and uptake abilities of the microsomal and mitochondrial fractions, while imipramine depressed the mitochondrial calcium uptake activity only at concentrations of 80 μm or higher. The mitochondrial respiratory and oxidative phosphorylation activities were depressed at high concentrations of these drugs. Since different membrane systems have been considered to be involved in the regulation of heart function and metabolism, the observed decreases in ATPase and calcium-accumulating activities of the heart subcellular membranes may represent one of the molecular mechanisms for the cardiodepressant actions of chlorpromazine and imipramine.     

Effect of papaverine on Ca2+ uptake by a mitochondrial fraction isolated from rat uterine smooth muscle

A mitochondrial fraction was isolated from rat uterine smooth muscle. The effect of papaverine (3 × 10^?6, 10^?5, 3 × 10^?5, 10^?4 M) on Ca²⁺ accumulation by the fraction was studied. The effect of papaverine was changed by using different substrates for Ca²⁺ accumulation. Papaverine inhibited the Ca²⁺ accumulation supported by glutamate plus ATP concentration-dependently. The Ca²⁺ accumulation supported by ATP alone was also inhibited. On the other hand, the Ca²⁺ accumulation supported by succinate and ATP was increased by papaverine. The effect was not concentration-dependent; papaverine (3 × 10^?5 M) was the most effective concentration for the effect with a shorter incubation (2 min).