ADP-Ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation.

We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 micromol/L) GT(1B), GD(1), and GM(3). Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N-endoglycosidase F and to thermal inactivation at 45 degrees C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn(2+) and Cu(2+) ions; the inhibition by Zn(2+) was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn(2+) stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-trans retinoic acid (atRA), and panagonist 9-cis-retinoic acid-upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)(2)-Vitamin D(3) (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED(50) congruent with 56 pmol/L). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3'-triiodothyronine (T(3)) to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from "classic" CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn(2+) and Cu(2+); (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T(3) in vitro; and (3) administration of atRA and T(3) increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.

gamma-L-glutamyl-L-DOPA inhibits Na(+)-phosphate cotransport across renal brush border membranes and increases renal excretion of phosphate.

BACKGROUND: For treatment of phosphate (Pi) overload in various pathophysiological states, an agent that selectively increases renal Pi excretion would be of major value. Previously, we have shown that dopamine (DA) inhibits Na(+)-Pi cotransport in renal epithelia. However, the administration of DA or its immediate precursor L-DOPA increases DA in multiple tissues. Synthetic dipeptide gamma-L-glutamyl-L-DOPA (gludopa) can serve as an inactive precursor (pro-pro-drug) of DA. This study tested the hypothesis that, because of the unique colocalization of gamma-glutamyltransferase (gamma-GT), aromatic amino acid decarboxylase, Na(+)-Pi cotransporter, and Na(+)-L-DOPA cotransporter in brush border membrane (BBM) of proximal tubular cells, gludopa may elicit phosphaturia by action of DA generated within the kidney. METHODS: Thyroparathyrectomized rats were given placebo, or gludopa, or gludopa + gamma-GT inhibitor acivicin. Urinary excretion of Pi, Ca2+, Na+, K+, DA, cAMP, and cGMP was determined, and Na(+)-Pi cotransport was measured in BBM prepared from kidneys of rats at the end of the experiment. RESULTS: The administration of gludopa resulted in: (a) an inhibition of Na(+)-Pi cotransport, but not cotransport of Na(+)-proline and Na(+)-alanine in BBM; (b) an increase (+300%) of fractional excretion (FE) of Pi and a drop (-35%) of plasma Pi, whereas the plasma levels and FEs of Ca2+, Na+, and K+ were unchanged; (c) an increase in urinary excretion of cAMP. but not cGMP; (d) a 1000-fold increase of urinary excretion of DA, without a change in excretion of norepinephrine; and (e) an incubation of gludopa with BBM in vitro, which caused a release of L-DOPA, and the in vivo administration of acivicin, which blocked actions of gludopa to inhibit Na(+)-Pi cotransport and to increase urinary excretions of Pi and DA. CONCLUSIONS: We conclude that colocalization of enzymes of biotransformation, BBM transporters, and the autocrine/paracrine DA system in cells of proximal tubules constitutes a cellular basis for the potent and specific phosphaturic action of gludopa.

Retinoic acid and triiodothyronine stimulate ADP-ribosyl cyclase activity in rat vascular smooth muscle cells.

Cyclic ADP-ribose (cADPR) is a nucleotide synthesized from beta-NAD- that can trigger or facilitate Ca2+-release through ryanodine-channels. We investigated the synthesis of cADPR (ADPR-cyclase activity) in cultured vascular smooth muscle cells (VSMC) from rat aorta in response to incubation with all-trans-retinoic acid (RA), 3,3',5'-triiodothyronine (T3), cortisol, beta-estradiol and 1-dehydrotestosterone. Only RA and T3 caused concentration-dependent (10(-9)-10(-6) M) stimulation of ADPR-cyclase activity in VSMC. Maximum stimulatory responses to RA (+100%) and T3 (+40%) were additive and the stimulatory effects of both hormones on ADPR-cyclase were due to an increase in Vmax without changes in the apparent Km. These observations indicate that in VSMC synthesis of cADPR can be upregulated by RA and T3. We propose that some of the actions of RA on VSMC such as enhancement of contractile competence, differentiation, and anti-proliferative effects might be elicited, at least in part, via upregulation of the cADPR/Ca2+-release signaling system.

Pleiotropic upregulation of Na(+)-dependent cotransporters by retinoic acid in opossum kidney cells.

All-trans-retinoic acid (atRA) is a regulator of cellular growth and differentiation. We investigated whether atRA can upregulate Na(+)-dependent cotransporters in opossum kidney (OK) cells and thus increase uptake from tubular fluid of several solutes needed for growth during early stages of ontogenesis. In OK cells, incubation with atRA for 24 h increased the Na+ gradient-dependent cotransports of phosphate, L-proline, L-glutamic acid, and SO(4)2- by a similar degree (approximately 40%) that was prevented by pretreatment with actinomycin D. In contrast, activities of other Na(+)-dependent transporters, Na(+)-K(+)-adenosinetriphosphatase, gamma-glutamyltranspeptidase, and leucine aminopeptidase, were unchanged by atRA. Cell proliferation determined by [3H]thymidine incorporation was not increased by atRA. The stimulatory effects of atRA and phosphate deprivation on Na(+)-Pi cotransport demonstrated additivity, whereas the combination of atRA and 3,5,3'-triiodothyronine did not. atRA stimulated Na(+)-Pi cotransport in LLC-PK1 cells with an analogous time course and to a similar extent as observed in OK cells. We conclude that atRA stimulates several Na(+)-dependent cotransporters via a genomic mechanism and may represent a synchronous adaptation to nutritional requirements of early phases of ontogenesis.

Opposite paracrine effects of 5-HT and dopamine on Na(+)-Pi cotransport in opossum kidney cells.

Serotonin (5-HT) was recently reported to inhibit cAMP generation in oppossum (OK) cells. We thus investigated the effects of 5-HT upon the Na(+)-Pi cotransport in cultured OK cells and its interactions with dopamine. Incubation of OK cells with 1 nM-10 microM 5-HT resulted in dose-dependent stimulation of Na(+)-Pi contransport (ED50 approximately equal to 8 nM) and also counteracted inhibition of Na(+)-Pi cotransport elicited by dopamine. Pre-incubation with 5-HT decreased cAMP accumulation elicited by forskolin or dopamine and pre-treatment with pertussis toxin abolished both the inhibitory effect of 5-HT upon cAMP levels and stimulation of Na(+)-Pi cotransport. Incubation of OK cells with the 5-HT precursor 5-hydroxytryptophan resulted in time- and dose-dependent accumulation of 5-HT in the medium that also elicited an increase in Na(+)-Pi cotransport. Both the effects of 5-HT and dopamine on Na(+)-Pi cotransport were prevented by carbidopa. The stimulatory effect of 5-HT was specific for the Na(+)-Pi cotransport system since no effects were observed on Na(+)-alanine cotransport. The results indicate that 5-HT stimulates Na(+)-Pi cotransport at least in part via inhibition of cAMP accumulation. We propose that 5-HT and dopamine have opposite actions as paracrine/autocrine regulators of Na(+)-Pi cotransport via opposite effects upon cAMP formation.

Effect of estrogen upon cyclic ADP ribose metabolism: beta-estradiol stimulates ADP ribosyl cyclase in rat uterus.

Cyclic ADP ribose (cADPR) has been shown to trigger Ca2+ release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all-trans-retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of beta-estradiol (E2), an archetypal steroid superfamily hormone, upon its major target organ, uterus, in vivo. Administration of E2 to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an approximately Delta + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E2-stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E2 was due to the increase of VMAX without change in Km. Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E2 (0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E2-treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E2, via action to its nuclear receptors in vivo, increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca2+ release pathway.

Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

The Ca(2+)-transporting ATPases of rabbit and trout exhibit different pH- and temperature-dependences.

The phosphorylation of the trout sarcoplasmic-reticulum Ca(2+)-ATPase by Pi differs in its temperature- and pH-dependence from the rabbit ATPase. In the trout enzyme, the apparent affinity for Pi and maximum phosphoenzyme values do not vary over a pH and temperature ranges that have a pronounced effect on the rabbit enzyme. The lack of temperature-dependence for phosphorylation is observed at pH 6.8. At pH 8.0, the temperature profile for phosphorylation of the trout enzyme resembles that of the rabbit at pH 6.8. The rabbit ATPase is no longer phosphorylated by Pi after solubilization with the detergent C12E9. In contrast, the trout enzyme can be phosphorylated by Pi after solubilization with C12E9, and the same levels of phosphoenzyme were obtained with the soluble and membrane-bound ATPase at both 0 degrees and 25 degrees C. In the range of 0-20 degrees C, the rates of ATP synthesis and of Ca2+ uptake by the trout ATPase are less temperature-dependent than for the rabbit enzyme. However, both isoenzymes catalyse ATP hydrolysis with similar temperature-dependences. The results raise the possibility that protonation of specific amino acid residues may contribute to the lack of temperature-dependence for phosphorylation of the trout Ca(2+)-ATPase.