Aldosterone induces ras methylation in A6 epithelia.

Aldosterone increases Na(+) reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 +/- 0.60 to 20.1 +/- 0.80 methyl ester cpm/microg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 +/- 0.64 in aldosterone-depleted cells to 31.8 +/- 2.60 methyl ester cpm/microg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S-trans, trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na(+) channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na(+) transport; 2) one target for methylation is p21(ras); and 3) inhibition of Ras expression or Ras methylation inhibits Na(+) channel activity.

S-adenosyl-L-homocysteine hydrolase regulates aldosterone-induced Na+ transport.

Aldosterone-induced Na+ reabsorption, in part, is regulated by a critical methyl esterification; however, the signal transduction pathway regulating this methylation remains unclear. The A6 cell line was used as a model epithelia to investigate regulation of aldosterone-induced Na+ transport by S-adenosyl-L-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolize S-adenosyl-L-homocysteine (SAH), an end product inhibitor of methyl esterification. Sodium reabsorption was decreased within 2 h by 3-deazaadenosine, a competitive inhibitor of SAHHase, with a half inhibitory concentration between 40 and 50 microM. Aldosterone increased SAH catabolism by activating SAHHase. Increased SAH catabolism was associated with a concomitant increase in S-adenosylmethionine catabolism. Moreover, SAH decreased substrate methylation. Antisense oligonucleotide complementary to SAHHase mRNA decreased SAHHase activity and Na+ current by approximately 50%. Overexpression of SAHHase increased SAHHase activity and dependent substrate methyl esterification. Whereas basal Na+ current was not affected by overexpression of SAHHase, aldosterone-induced current in SAHHase-overexpressing cells was significantly potentiated. These results demonstrate that aldosterone induction of SAHHase activity is necessary for a concomitant relief of the methylation reaction from end product inhibition by SAH and the subsequent increase in Na+ reabsorption. Thus, regulation of SAHHase activity is a control point for aldosterone signal transduction, but SAHHase is not an aldosterone-induced protein.

Cytoplasmic calcium buffer capacity determined with Nitr-5 and DM-nitrophen.

We have examined intracellular calcium buffer capacity of cytoplasm from the giant axon of the marine invertebrate Myxicola infundibulum by photolytically releasing calcium from 'caged' compounds, while monitoring free calcium, [Ca2+], with Ca-sensing electrodes. In cytoplasm containing intact organelles, two features of the [Ca2+] response were seen upon light exposure: an initial spike from basal [Ca2+], followed by a slower phase recovery. Both the amplitude of the spike in [Ca2+] and the recovery were reduced by removal of MgATP. If organelles were removed from the cytoplasm, light exposure caused only a step-like change in [Ca2+] with no recovery. Apparent buffer capacities (delta bound Ca/delta free Ca) were unaffected by changing pH from 7.0 to 7.5; however, raising basal free calcium above 3 microM significantly reduced this parameter. The buffer capacity measured after the initial spike varied by as much as an order of magnitude from one giant axon to another but averaged approximately 50 in the absence and approximately 100 in the presence of 1 mM MgATP for [Ca2+] below 3 microM.

Calcium diffusion coefficient in Myxicola axoplasm.

Calcium diffusion coefficients were measured in Myxicola axoplasm and in agar controls by two independent techniques: one utilizing 45Ca, and one utilizing Ca-specific mini-electrodes. The lowest value, approximately 0.1 x 10(-6) cm2.s-1, was measured, using the mini-electrode technique, in axoplasm with intact Ca-sequestering organelles. With ATP-depleted axoplasm, diffusion coefficients of 0.5-2 x 10(-6) cm2.s-1 were obtained by both isotope and mini-electrode techniques. In organelle-free axoplasm with a protein concentration roughly half that in the intact axoplasm, diffusion coefficients of 1.4-3 x 10(-6) cm2.s-1 were measured at 0.7 microM Ca and 7 x 10(-6) cm2.s-1 at 3-5 microM Ca. When compared with measurements of the calcium buffering capacity [Al-Baldawi NF. Abercrombie RF. (1995) Cytoplasmic Ca buffer capacity determined with Nitr-5 and DM-nitrophen. Cell Calcium, 17, 409-422], these diffusion coefficients require that part of the buffer capacity be located on mobile Ca-binding sites.

Calcium accumulation by organelles within Myxicola axoplasm.

1. 45Ca2+ accumulation into inulin-inaccessible compartments within cytoplasm from the giant axon of Myxicola infundibulum was measured as a function of free calcium, pH, and time. Accumulation reached a maximum after 1 h and remained stable for at least 3 h. 2. At 0.5, 5, and 50 microM [Ca2+], in the presence of 1 mM ATP or 5 mM succinate, steady-state calcium uptake had a bell-shaped dependence on pH with a maximum near pH 7. Uptake was abolished by the proton uncoupling reagent carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 4 micrograms ml-1). 3. Uptake of the membrane permeant cation, [14C]-tetraphenylphosphonium (TPP+), also had a bell-shaped dependence on pH with a maximum pH approximately 7, suggesting a pH dependence of the electrical potential of a membrane enclosed cytoplasmic compartment. Cyanide (2 mM) inhibited TPP+ uptake. 4. Inositol 1,4,5-trisphosphate (IP3, 10 microM), reduced steady-state calcium accumulation by 20-22% at 0.5 microM free calcium, pH 7 (P < 0.01, n = 16) and at 5 microM free calcium, pH 8 (P < 0.0005, n = 35). No effects of IP3 were found at other pH or calcium concentrations. 5. Neither guanosine 5'-triphosphate (GTP) nor inositol 1,3,4,5-tetrakisphosphate (IP4) had an effect on calcium uptake (5 microM [Ca2+], pH 8). 6. At 0.5 microM free calcium; vanadate (10 microM) inhibited 20-30%, of the 45Ca2+ accumulation, thapsigargin (33 nM) inhibited 20-30%, and cyanide (2 mM) plus oligomycin B (2 micrograms ml-1), or valinomycin (1 microM), inhibited 70-80%. The fraction of uptake sensitive to thapsigargin fell as the free calcium increased; however, the sensitivity of uptake to cyanide plus oligomycin B was approximately 80% for 0.5, 5.0, and 50 microM [Ca2+]. 7. Thapsigargin had no additional inhibiting effect in the presence of cyanide plus oligomycin B. IP3 had no effect in the presence of cyanide plus oligomycin B or other mitochondrial inhibitors. 8. Results suggest the presence of both mitochondrial (70-80%) and non-mitochondrial (20-30%) calcium pools in this system (at 0.5-5.0 microM Ca2+). The apparent non-mitochondrial uptake (sensitive to thapsigargin, or IP3) is not detectable in the presence of mitochondrial inhibitors. We interpret these results as evidence of functional communication between mitochondrial and non-mitochondrial calcium stores.

Cytoplasmic hydrogen ion diffusion coefficient.

The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient.

Properties of calcium binding by Myxicola axoplasmic protein.

The 45Ca2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca2+ is attached to a site with an equilibrium dissociation constant of 7.7 +/- 0.5 microM and a capacity of 4.4 +/- 0.2 mumol/g axoplasmic protein (n = 11). Addition of other cations--Cd2+, Mn2+, Al3+, Cu2+, Ba2+, and Zn2(+)--at concentrations up to 10 microM did not displace 0.2 microM 45Ca2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4 degrees C for up to 16 days with no appreciable change in the number of calcium sites. Ca2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4 degrees C to 37 degree C reduced the number of Ca2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ binding by Myxicola neurofilament proteins.

Titrimetric, 45Ca dialysis, and autoradiographic methods were used to examine how axoplasmic proteins from the giant neuron of the marine annelid Myxicola infundibulum bind calcium. Following the autoradiographic method of Maruyama et al., the 150-160 kD neurofilament subunits were identified as prominent intracellular Ca-binding peptides. Using equilibrium dialysis, extracts of axoplasmic proteins (greater than 50% neurofilament subunits) were examined in 300 mM KCl at different concentrations of free Ca and Mg, and at different pH. Axoplasmic proteins showed a high affinity Ca binding site (K1/2 3-6 microM, capacity 3-7 mumole g-1 protein) at pH 6.8 or pH 7.5. Changing the Mg concentration from 0 to 5 mM had no effect on the Ca binding. Elevating the dialysis pH from 7.0 to 9.0 reduced the apparent number of binding sites for Ca. Using microelectrodes to record the free Ca, microtitrations of axoplasmic proteins were completed by adding small amounts of CaCl2 to 100 microliters volumes of protein solutions. In a medium containing ionic constituents closely resembling those of the Myxicola axon, a Ca binding capacity of 5.0 mumole g-1 protein and a K1/2 of approximately 1 microM were measured.

Metabolism of the amino acid beta-pyrazol-1-ylalanine and its parent base pyrazole.

beta-Pyrazol-1-yl-DL-alanine, an uncommon amino acid from plants of the Cucurbitaceae, was fed to mice. Although pyrazole is known to affect the liver enzymes UDP-glucose dehydrogenase, UDP-glucuronyl transferase and UDP-glucuronic acid pyrophosphatase, and also depresses their liver glycogen concentrations, beta-pyrazol-1-ylalanine had no such effects. beta-Pyrazol-1-ylalanine could not be detected in the liver of the experimental animals but was present in the urine. No other change in urinary amino acid content was observed. Studies with [14C]-beta-pyrazol-1-yl-DL-alanine showed the administered amino acid was excreted over a 4-day period, 93% of the compound supplied was recovered. Similar recoveries were obtained with the L-enantiomer from cucumber seed. The metabolic inertness of beta-pyrazol-1-ylalanine was also apparent in experiments involving subcutaneous injection of this compound. Administration of pyrazole confirmed an earlier report of resultant increased activity of liver UDP-glucose dehydrogenase and UDP-glucuronyl transferase, and of the depression of activity of liver UDP-glucuronic acid pyrophosphatase. A concomitant 40% decrease in liver glycogen content was seen. The urine contained a novel metabolite, identified as a peptide conjugate of a pyrazole derivative. Mass spectrometry and p.m.r. spectroscopy indicate that this derivative is 3,4,4-trimethyl-5-pyrazolone. The amino acid constituents are aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine and leucine. The urine of mice receiving pyrazole contained less free glycine and alanine than controls. From the results, it is concluded that pyrazole is not a catabolite of dietary beta-pyrazol-1-ylalanine but to the contrary, the amino acid is essentially excreted unchanged. Formation of 3,4,4-trimethyl-5-pyrazolone from pyrazole would imply C-methylation, a process that has not been previously observed in a mammalian detoxication context.