Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

The effect of local anesthetics on calcium transport across the rat and turtle small intestine

Procaine at different concentrations enhanced significantly (P less than 0.01) calcium accumulation in rat intestinal cells, whereas the same concentrations of procaine inhibited significantly (P less than 0.01) calcium uptake by the turtle small intestine. Unidirectional calcium influx across the rat small intestine was significantly enhanced (P less than 0.001) by the presence of procaine in the preincubation medium. However, procaine had no effect on calcium influx across the turtle intestinal cells. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with procaine in both animals.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cysteamine-induced duodenal ulcer: effects on calcium absorption across rat duodenum

The effect of cysteamine-induced duodenal ulcers on calcium transport across rat duodenum was investigated. Intracellular calcium accumulation measured after 24 hr and 3 days of cysteamine injection showed a significant increase (P less than 0.001) in the duodenal strips isolated after 3 days with no change noticed in those isolated after 24 hr, although the morphological changes in both were very similar. The relationship between increasing calcium concentration in the incubation medium and intracellular calcium concentration is a saturable process that conforms to the Michaelis-Menten type of kinetics. The average maximal flux (Vmax) increased from 8.93 nmole/hr-gdw in normal to 12.5 nmole/hr-gdw in 3-day-ulcerated rats, with no apparent change in the Michaelis constant (Kt) (0.8 mM). Unidirectional influx of calcium across the mucosal membrane was significantly increased (P less than 0.001) in 3-day-ulcerated duodenum suggesting that the increase in calcium transport could be due to the activation of the active step at the mucosal border.     

The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.     

Characterization of P2 receptors in thymic epithelial cells.

The presence of P2 receptors was investigated in three distinct preparations of murine thymic epithelial cells (TEC): 2BH4 murine cell line, IT45-R1 rat cell line, and a primary murine cell derived from the Nurse cell lympho-epithelial complex. In all preparations, application of ATP to the extracellular milieu triggered intracellular calcium signals indicating the presence of P2 receptor(s) in these cells. After an initial peak of calcium concentration, a plateau phase that could last more than 10 min was frequently observed. Ion replacement and channel blockage experiments indicated that the initial peak was associated with the release of calcium from intracellular stores, while the plateau phase was associated with an influx from the extracellular medium. ATP and UTP induced similar calcium signals, suggesting the presence of P2Y2 receptors in all three cell types. The murine 2BH4 cells also expressed P2X7/P2Z receptor, since under exposure to millimolar concentrations of ATP, a continuous rise in intracellular calcium concentration was observed and their plasma membranes became permeabilized to the fluorescent dyes Lucifer yellow and ethidium bromide. In addition, this permeabilization phenomenon was blocked by the P2Z-specific antagonist, oxidized ATP. RT-PCR assays confirmed the presence of mRNAs for the P2Y2 molecule in all TEC, while mRNA for the P2X7 molecule was detected only in 2BH4 cells. Our data indicate that P2Y2 purinergic receptors are widely expressed by thymic epithelial cells, whereas the expression of the P2X7 receptor appears to be more restricted, raising the possibility that its expression is related only to a particular epithelial microenvironment within/the thymus.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis

1. We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of 45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes). 2. Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 +/- 0.23- and 3.71 +/- 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P less than or equal to 0.1). 3. Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 +/- 5- and 20 +/- 3-fold, respectively, and A23187-induced secretion was increased 1.3 +/- 0.4- and 1.3 +/- 0.1-fold (P less than or equal to 0.01 for both ionophores). 4. The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P less than or equal to 0.01 for sodium greater than or equal to 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1. 5. The rate of 45Ca efflux from neurosecretosomes showed 2.7 +/- 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P less than or equal to 0.01). 6. Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.     

The cellular transport of calcium in rat liver

The bidirectional transport of calcium in rat liver was studied using slices labeled with Ca⁴⁷ in a closed two compartment system. Steady-state conditions were observed with influx and efflux transfer coefficients of 0.070 and 0.018 per minute, respectively. The rapidly exchanging cell fraction of calcium existed at a concentration three times higher than the average cell concentration of calcium and occupied cell loci comprising less than 25% of the cell mass, suggesting that calcium associated with the cell membranes, nuclei, and mitochondria participated in the rapidly exchanging fraction. At pH 7.4 and 377deg;C, the influx transfer coefficient was 25% above the steady-state condition and accumulation of calcium by the slices occurred. Studies of the effects of varied physical and chemical conditions revealed that the influx transfer coefficient was increased by elevated pH, strontium, certain metabolic inhibitors, and 2 mM concentrations of cyclic adenosinemonophosphate and adenosinetriphosphate. The influx transfer coefficient was decreased by reduced temperature, decreased pH, magnesium, and 10 mM adenosinetriphosphate. The efflux transfer coefficient was increased by elevated pH, strontium, iodoacetate, and adenosinetriphosphate, and was decreased by reduced temperature and by N-ethylmaleimide. These data support the thesis that cell transport of calcium is accomplished by the attachment of calcium atoms to the cell surface and transport through the plasma membrane bound to either specific carriers or to membrane constituents. Conditions which change the affinities, capacities, and mobilities of plasma membrane ligands that bind calcium or cause extracellular chelation of calcium are capable of altering the rate of calcium transport.     

Potentiating effect of NH4Cl on vasoconstriction in rat aorta.

The effect on the vasocontractile response of pretreatment with NH4Cl at a concentration (10 mM) that made almost no change in the resting tension was investigated using aortic strips from rats. NH4Cl pretreatment for 10 min significantly potentiated strip contractions induced by KCl (less than or equal to 30 mM), BAY K 8644 (0.1 microM) and phenylephrine (0.01 microM). This potentiating action of NH4Cl was eliminated in presence of nifedipine (1 microM). KCl (14.7 mM)-stimulated 45Ca uptake in rat aorta was significantly potentiated by pretreatment with NH4Cl (10 mM) for 10 min, but this NH4Cl effect was also eliminated in the presence of nifedipine. These results suggest that NH4Cl potentiates contractions induced by KCl and agonists in rat aorta by facilitating calcium influx through the nifedipine-sensitive calcium channel.     

牛血清白蛋白耦联皮质酮可以抑制精氨酸加压素的释放

本实验利用高体孵育脑薄片和放射免疫测定精氨酸加压素（ＡＶＰ）的方法,初步探讨了牛血清白蛋白耦联皮质酮（Ｂ－ＢＳＡ,不易进入细胞内）对大鼠下丘脑薄片（含室旁核和视上核）释放ＡＶＰ的影响和可能机制。结果：（１）Ｂ－ＢＳＡ（１０－７─１０－４ｍｏｌ／Ｌ）在２０ｍｉｎ内对大鼠下丘脑薄片ＡＶＰ的释放具有明显的抑制性效应,且呈剂量一效应关系;（２）ＲＵ４８６（１０－４─１０－３ｍｏｌ／Ｌ）能部分地阻断Ｂ－ＢＳＡ的抑制效应;（３）Ｂ－ＢＳＡ的抑制效应随孵育液中Ｃａ２＋浓度的升高而明显增强;（４）在有新毒素（１０－３─１０－２ｍｏｌ／Ｌ）存在的情况下,Ｂ－ＢＳＡ的抑制效应显著增强。上述结果表明糖皮质激素在未进入细胞内的情况下亦可抑制大鼠下丘脑薄片释放ＡＶＰ。此作用发生在细胞膜水平上,由非基因组机制所介导,可能是影响Ｃａ２＋跨细胞膜流动的结果。     

Effect of enzymatic treatment on calcium absorption by small intestine: a comparative study between rat and rabbit transport mechanisms

Calcium absorption by the small intestine of rat and rabbit reached steady state after 60 min of incubation with intracellular to extracellular ratio of 2.0. Trypsin and neuraminidase significantly inhibited (P less than 0.05) calcium accumulation in rat small intestine. These enzymes showed no significant effect (P greater than 0.05) on calcium transport across rabbit small intestine. The inhibitory action of trypsin and neuraminidase on calcium accumulation by the rat small intestine does not involve the influx of calcium into the intestinal cells.     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease. Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (delta +480%; p less than 0.001) compared to sham-operated controls. In vivo, indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (delta -34%; p less than 0.05) and 90 min (delta -47%; p less than 0.05). In vitro renin release from renal cortical slices of ADX rats was also significantly greater (delta +130%; p less than 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 micrograms/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values. Prostaglandin E2 (PGE2) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE2 synthesis in glomeruli microdissected from ADX rats was significantly increased (delta +110%; p less than 0.001) compared to controls. PGE2 synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10(-6) M) decreased PGE2 synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices. These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

Kinetics of Tryptophan Influx into Brain Slices Depleted of Sodium and Loaded with l-Histidine

Abstract: The kinetics of tryptophan influx were studied with rat brain slices preloaded with l -histidine and/or depleted of sodium ions. The best fits of the data (velocity of influx versus tryptophan concentration) were computed by use of a model consisting of a saturable (Michaelis-Menten type) and an unsaturable (diffusional) component with an iterative nonlinear regression analysis. Sodium depletion of the slices reduced the maximal velocity of saturable influx. In histidine-preloaded slices, depleted or not depleted of sodium ions, the most marked alteration again occurred in the maximal velocity, which more than doubled. Slices preloaded with histidine contained greatly elevated levels of glutamine and histidine, which may have stimulated the influx by exchange with extracellular tryptophan even in the absence of sodium ions. The maximal velocity was higher with increasing concentration of large neutral amino acids in slices at the start of the influx measurements. The influx of tryptophan in brain cells is apparently modified by changes in the intracellular amino acid pool, which, when increased, also counteracts the effect of sodium depletion on the tryptophan influx.     

Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells

Cholera enterotoxin (CT), at an optimal concentration of 2.38 X 10(-10) M, stimulated calcium uptake (P less than 0.01) and cyclic AMP accumulation (P less than 0.02) in cultured rat basophilic leukemia cells. No significant effect of CT on calcium release or cyclic GMP accumulation was detected. Pharmacologic and chemical agents which block calcium uptake or prostaglandin synthesis antagonized the effect of CT.     

Beta-adrenergic stimulation evokes a rapid, Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca2+ fluxes in mouse kidney cortex

The beta-adrenergic agonist 1-isoproterenol evokes an acute (less than 5 min) stimulation of endocytosis, hexose transport and amino acid transport, measured by the temperature-sensitive uptake of HRP, 3H-DG and 14C-AIB, in mouse kidney cortex slices. This stimulation is concentration dependent and is maximal at 10(-8)-10(-7) M isoproterenol. Peroxidase cytochemistry showed that the hormonal increase in HRP uptake is confined to proximal tubules. The rapid membrane response is abolished in a calcium-free medium and by the beta-adrenergic antagonist propranolol, indicating Ca2+- and beta-adrenoreceptor-dependence. Isoproterenol (1 microM) rapidly (less than 30 sec) stimulates the influx and efflux of 45Ca in cortex slices. Isoproterenol also decreased mitochondrial 45Ca and increased soluble 45Ca. These results indicate that beta-adrenergic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular (mitochondrial) calcium. An increase in cytosolic Ca2+ concentration appears to be the regulatory signal for these membrane transport processes.     

Role of calcium in the potentiating effect of phorbol ester on KCl-induced vasocontraction

The mechanism of the potentiating effect of phorbol ester on potassium-induced contraction in rat aorta was investigated. The contractile response to KCl in the medium containing 0.5 mM CaCl2 was significantly increased by pretreatment with 10(-8) M phorbol 12-myristate 13-acetate (PMA), but not with 10(-7) M 4 alpha-phorbol. The dose-response curve to calcium in 30 mM KCl-induced contraction was shifted to the left by PMA pretreatment and the EC50 value (the concentration producing a half maximal response) of calcium was significantly lower in aorta pretreated with PMA than in the control. On the other hand, calcium influx stimulated by 30 mM KCl was not changed by PMA pretreatment. Both the contractile response and the corresponding calcium influx induced by 30 mM KCl were abolished by preincubation with 10(-6) M verapamil for 45 min. These results suggest that activation of protein kinase C potentiates the contractile response to KCl by increasing the sensitivity of the intracellular contractile apparatus for calcium.     

Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells

When aequorin-loaded glomerulosa cells were incubated in isotonic Na2+-free medium containing N-methyl-D-glucamine instead of NaCl, there was an increase in cytoplasmic free calcium concentration, [Ca2+] c, which was not observed when extracellular calcium concentration was reduced to 1 microM. Upon removal of extracellular sodium, there was nearly five-fold increase in fractional efflux ratio of calcium. The reduction of extracellular sodium resulted in a stimulation of calcium influx rate, the magnitude of which was dependent on extracellular sodium concentration. Similar stimulation of calcium influx was observed when extracellular sodium was replaced with lithium. Nitrendipine did not affect the calcium influx induced by the reduction of extracellular sodium while a derivative of amiloride 3',4'-dichlorobenzamil, which inhibits Na-Ca exchange, attenuated calcium influx observed in sodium-free medium. These results indicate that removal of extracellular sodium leads to an increase in [Ca2+] c by stimulating calcium influx and that calcium enters the cell via Na-Ca exchanger.