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K W Snowdowne

Publications and source records attributed to K W Snowdowne.

At least 19 recordsLinked to original sources
PubMed1999

Angiotensin-II increases cytoplasmic calcium, cell number and total DNA for human periodontal ligamental cells in vitro.

The identification and the development of potential growth factors for the cells of the periodontal ligament is an important and novel stratagem for the regeneration of tissue destroyed by periodontal pathogens. The problem is that growth factors are difficult to synthesize, very immunogenic and expensive. The purpose of our study was to determine if angiotensin-II, a naturally occurring octo-peptide with growth-factor like properties in cardiac muscle, could also stimulate cultured human periodontal ligament cells. If angiotensin-II does have growth factor properties, then it should be able to increase cytoplasmic calcium (a secondary messenger associated with mitogenesis) and to increase fibroblast proliferation. Fura-2 fluorescence and aequorin photometry were used to follow changes in cytoplasmic calcium. Cytometry and total amount of DNA were used to measure proliferation evoked by angiotensin-II. We found that angiotensin-II increased cytoplasmic calcium, the total amount of DNA and cell number, suggesting that angiotensin-II has mitogenic properties for cells isolated from human periodontal ligament.

Aequorin↗
PubMed1993

Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells.

We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

3T3 Cells↗
PubMed1992

pHi controls cytoplasmic calcium in rat parotid cells.

The goal of this investigation was to determine if cytoplasmic pH (pHi) modulated the basal level of the concentration of calcium ions in the cytoplasm (Cai) in rat parotid cells. We investigated the effects of various experimental manipulations on both pHi and Cai as measured with BCECF and the calcium photoprotein aequorin, respectively. We found that various experimental manipulations that increased pHi, such as exposure of the cells to NH4Cl, a decrease of the partial pressure of CO2 or an increase in extracellular pH in the presence of nigericin invariably increased Cai. Moreover, experimental manipulations which lowered Cai, such as a reduction of extracellular [NaHCO3] or the removal of loaded NH4 invariably decreased Cai. Thus pHi and Cai are directly related in parotid cells. Since recent studies have shown that Cai directly influences pHi, we suggest that Cai-handling and pHi-handling are tightly linked in parotid cells.

Aequorin↗
PubMed1992

Prolactin secretion in anterior pituitary cells: effect of eicosanoids.

Ovariectomized Fischer 344 rats were implanted with silastic capsules containing estradiol for approximately 4 weeks to increase the number of lactotrophs in the anterior pituitary gland. Anterior pituitary cells were enzymatically dispersed and cultured for 1 day and challenged with eicosanoids or other prolactin (PRL) secretagogues. The isolated pituitary cells from estradiol-pretreated animals exhibited an increase in the ability to secrete PRL in the presence of maximally effective concentrations of thyrotropin releasing hormone (TRH), arachidonic acid or 5,6-epoxyeicosatrienoic acid (5,6-EET). Anterior pituitary cells from estradiol-pretreated animals also showed an increased activity of cytochrome P-450. The possible involvement of cytochrome P-450 in PRL secretion from anterior pituitary animal cells isolated from animals pretreated with estradiol was shown by the fact that these anterior pituitary cells increased the synthesis of 5,6-EET, a potent PRL releasing agent. TRH and 5,6-EET increased the mobilization of both cyclic AMP and cytoplasmic calcium to about the same extent. The data suggest that cytochrome P-450 is important in the release of PRL from anterior pituitary cells isolated from estradiol-pretreated Fischer 344 rats and that a product of cytochrome P-450-catalyzed epoxidation of arachidonic acid, 5,6-EET, plays a significant role in the release of PRL.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Eicosanoids evoke the release of amylase and increase cytoplasmic calcium in rat parotid cells.

The effects of various eicosanoids on cytoplasmic calcium and the release of amylase were examined in isolated rat parotid cells. Arachidonate and several of its metabolites increased amylase release and elevated cytoplasmic calcium. Melittin, a stimulator of arachidonate mobilization, and lyso-phosphatidylcholine also released amylase and elevated calcium. These results suggest that the metabolites of arachidonate may have an important role in amylase secretion.

Amylases↗
PubMed1989

Evidence for a sodium/calcium exchanger and voltage-dependent calcium channels in adipocytes.

The objective of these studies is to identify and characterize Ca2+-transport systems that may be of potential importance in the action of Ca2+-mobilizing hormones in the adipocyte. Using the Ca2+-sensitive photoprotein, aequorin, [Ca2+]i was estimated to be 0.15 microM, assuming an intracellular [Mg2+] of 1 mM. Substitution of Na+ with choline+ caused a transient increase in [Ca2+]i which was inversely related to extracellular [Na+], consistent with operation of a mediated Na+-Ca2+ exchange system. The stoichiometry was 3Na+:Ca2+. Elevation of extracellular K+ caused an increase in [Ca2+]i that was blocked by the Ca2+ channel antagonist, diltiazem, by omitting extracellular Ca2+, or by substituting Sr2+ for Ca2+. These findings indicate the presence of an Na+-Ca2+ exchanger and voltage-sensitive Ca2+ channels in adipocytes.

Adipose Tissue↗
PubMed1987

Intracellular messenger for action of angiotensin II on fluid transport in rabbit proximal tubule.

These studies were designed to examine the cellular messenger that mediates the action of angiotensin II on fluid transport (Jv) in the rabbit proximal tubule. We measured the effects of angiotensin II on Jv, activation of adenylate cyclase, and the concentration of cytosolic free calcium (Cai) in the rabbit proximal tubule. In nine rabbit proximal convoluted tubules (PCT), angiotensin II, 10(-8) M and 10(-6) M, decreased Jv by 18 and 25%, P less than 0.05. In eleven rabbit proximal straight tubules (PST), 10(-8) and 10(-6) M angiotensin II decreased Jv by 20 and 23%, P less than 0.02. Angiotensin II did not affect lumen-to-bath phosphate fluxes in PCT or PST, and it did not activate adenylate cyclase in PST. In a preparation of proximal tubules (PCT and PST) loaded with aequorin, angiotensin II, 10(-8) and 10(-6) M, transiently increased Cai by 13 and 32%, P less than 0.001. We propose that Cai surges, activated by angiotensin II, are part of a cellular message that inhibits Jv in the rabbit proximal tubule.

Adenylyl Cyclases↗
PubMed1986

The effect of stretch on sarcoplasmic free calcium of frog skeletal muscle at rest.

The concentration of free calcium in the sarcoplasm of a resting frog skeletal muscle is increased by stretch. The magnitude of the rise in free calcium increased with the degree of stretch and the ambient temperature and it was enhanced by caffeine. This phenomenon might play a role in the twitch potentiation and enhanced metabolic rate evoked by stretch.

Animals↗
PubMed1986

Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am.

Fura-2-am, the pentaester precursor of the fluorescent Ca(2+) indicator fura-2, is modified when it is exposed to isolated skeletal muscle sarcoplasmic reticulum vesicles. The modified fura-2-am has enhanced fluorescence, is not sensitive to Ca(2+), and is partially bound to the SR membrane. The isolated product is further converted into fura-2 by esterase. It is suggested that the SR-induced modification is a selective enzymatic hydrolysis of only some of the five ester moieties on fura-2-am. A structure is proposed to account for the results. The potential for this effect of SR on fura-2-am to cause complications in measurements of in vivo intracellular free [Ca(2+)] is noted.

Animals↗
PubMed1986

Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing.

The concentration of cytosolic-free calcium (Ca2+i) was determined with aequorin in RAW-264 macrophage-like cells activated in vitro for tumor cell killing with lymphokine (LK) and lipopolysaccharide (LPS). Treatments of these cells with optimal doses of stimulants, which evoked the development of cytolytic activity, also induced a rise in their Ca2+i. No rise in Ca2+i could be observed under treatments which failed to activate cells. The presence of both stimulants was an absolute requirement for evoking cytolytic activity and also a rise in Ca2+i. There was an apparent parallelism between the rate of activation and the rate of rise in Ca2+i. Cells which slowly developed their cytolytic activity exhibited a slow rise in Ca2+i, while macrophages which acquired their cytolytic activity at the faster rate also showed a more rapid increase in Ca2+i. The development of cytolytic activity in RAW-264 macrophages was inhibited by two intracellular calcium antagonists, TMB-8 and ruthenium red. This inhibition could be reversed by high concentrations of extracellular calcium. TMB-8, at the concentrations which were effective in inhibiting the activation process, also completely blocked the associated rise in Ca2+i. These results suggested that Ca2+i might play a role in the mechanism of tumoricidal transformation of RAW-264 macrophages.

Aequorin↗
PubMed1986

A simple method for incorporating aequorin into mammalian cells.

A simple method for incorporating aequorin into mammalian cells to measure cytosolic ionized Ca2+ is described and compared with scrape loading and hypoosmotic treatment (HOST). The procedure consists of incubating the cells for 10 min and centrifuging them at 200 g for 30 s in the presence of aequorin. This method incorporates the same amount of photoprotein as scrape loading but 70% less than HOST. Cytosolic ionized Ca2+ has been measured in hepatocytes, kidney cells and tubules, macrophages, and cardiac myocytes loaded with aequorin by this new procedure.

Aequorin↗
PubMed1986

Cytosolic free calcium and DNA synthesis in BALB/c 3T3 cells: aequorin luminescence studies.

In order to investigate how growth factors stimulate DNA synthesis, we studied the relationship between early increases in the intracellular concentration of free calcium (Cai) and early mitogenic events (competence) in quiescent BALB/c 3T3 cells. Cai was measured by aequorin luminescence, and DNA synthesis was quantitated by autoradiography. While both serum (2%) and platelet-derived growth factor (2 U/ml) stimulated large increases in Cai without inducing competence, fibroblast growth factor (100-400 ng/ml) produced a large number of competent cells without causing a detectable increase in Cai. Thus, there was no correlation between the magnitude of Cai increase and subsequent competence for DNA synthesis.

Aequorin↗
PubMed1985

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells.

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.

Animals↗
PubMed1985

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells.

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.

Adenosine Triphosphate↗
PubMed1985

Measurement of cytosolic calcium with aequorin in dispersed rat ventricular cells.

The changes in the cytosolic concentration of free calcium of adult rat cardomyocytes were monitored using aequorin incorporated by hypoosmotic shock. The majority of the myocytes retained their rod-like appearance and their tolerance to a normal concentration of extracellular calcium. The aequorin signal was increased by depolarization, by an increase in extracellular calcium, by substitution of extracellular sodium with either choline or tetramethylammonium, by 20 mM NH4Cl or by hypoxia. In these myocytes, isoproterenol or the phosphodiesterase inhibitor MIX (3-isobutyl-l-methylxanthine) enhanced the ability of the cells to buffer calcium loads while the mitochondrial inhibitor FCCP (carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone), decreased their calcium buffering capacity.

Aequorin↗
PubMed1985

Subcontracture depolarizations increase sarcoplasmic ionized calcium in frog skeletal muscle.

The hypothesis that the sarcoplasmic concentration of ionized calcium (Cai) of frog skeletal muscle may control its rate of metabolism was examined by monitoring the changes in Cai due to perturbations that are known, from the work of previous investigators, to alter the rate of metabolism. Cai was measured with aequorin, which was microinjected into isolated fibers in sufficient quantity to detect basal Cai. When these fibers were exposed to 5-18 mM KCl, 75 mM RbCl2, or 100 mM CsCl2, there was an increase in the aequorin signal. The potassium-evoked increase in the aequorin signal was diminished by an increase in the extracellular concentration of Ca or by Ca-free media containing 3.6 mM Mg, Mn, Sr, or Zn but was enhanced by subcontracture concentrations of caffeine or media containing nitrate instead of chloride. These results are consistent with the hypothesis that Cai may control the rate of metabolism in frog skeletal muscle fibers.

Aequorin↗
PubMed1984

Regulation of hepatic glycogenolysis by glucagon in male and female rats. Role of cAMP and Ca2+ and interactions between epinephrine and glucagon.

The effects of 10(-10) to 10(-7) M glucagon on cAMP, phosphorylase a, cell calcium, and glucose production, and glucagon interactions with epinephrine were studied in isolated hepatocytes from adult male and female rats. At physiological concentrations (10(-10) - 10(-9) M), glucagon activated phosphorylase by increasing cAMP and not by raising the cytosolic free calcium. At supra-physiologic concentrations (and in the male only), glucagon slightly increased the cytosolic free calcium, the fractional efflux of calcium, and, after 2 h, decreased the cell calcium content. Exposure of hepatocytes to the simultaneous administration of 10(-9) M glucagon and 10(-7) M epinephrine resulted in a prolongation of the activation of phosphorylase a and a greater release of glucose from glycogen stores than exposure to either agonist alone. In the male, the effects of low concentrations of the two hormones on phosphorylase a activity were additive. Cytosolic free calcium was increased by 10(-6) M epinephrine from 280 to 500 nM while physiological concentrations of glucagon did not change it. In these intact cells, there was no evidence of an alpha 2-adrenergic inhibition of adenyl cyclase and no indication that cAMP depresses the rise in cell calcium induced by alpha-adrenergic stimuli.

Animals↗