Interactions of ANP and ANG II in tubular nephron acidification

(ANP, 1 µM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H+-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H+-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF. We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange. ANP (1 µM) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [Ca2+]i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension. The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 µM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times the basal level. In contrast, the addition of ANP (1 µM) to the cell suspension led to a very rapid 60% decrease in [Ca2+]i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i. These results may indicate a role of [Ca2+]i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II. The data are compatible with stimulation of Na+/H+ exchange by increases of [Ca2+]i in the lower range, and inhibition at high [Ca2+]i levels

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the<FONT FACE="Symbol"> a</font>-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension

Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD+-NADH redox potentials

The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c) In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a well-defined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca2+ except when the lactate to pyruvate ratio was equal to 1. The results of the present study demonstrate that stimulation of gluconeogenesis by glucagon depends on Ca2+. However, Ca2+ is only effective in helping gluconeogenesis stimulation by glucagon at highly negative redox potentials of the cytosolic NAD+-NADH system. The triple interdependence of glucagon-Ca2+-NAD+-NADH redox potential reveals highly complex interrelations that can only be partially understood at the present stage of knowledge

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß)2 association.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation

Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.

Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ± 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ± 2.4 and 6.7 ± 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ± 0.97 and 7.5 ± 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.