The role of sex steroids on cellular events involved in vascular disease.

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

Progesterone and 17 beta-estradiol acutely stimulate nitric oxide synthase activity in rat aorta and inhibit platelet aggregation.

The rapid non-genomic stimulatory action of progesterone (Pg) and estradiol (E2) on nitric oxide synthase (NOS) activity of endothelium intact aortic rings and its effect on platelet aggregation was investigated. First we measured the effect of the hormones on platelet aggregation when added to rat aortic strips (RAS) incubated in a PRP. RAS induced an antiaggregatory activity, which was enhanced by the presence of the hormones. The inhibitory action induced by the hormones was evoked in a dose dependent manner (10 pM-100 nM). These effects are specific for progesterone and 17-beta-estradiol, since either testosterone and 17-alpha-estradiol were devoid of activity. The hormones induced rapid responses, producing significant inhibition within 1 to 5 minutes of hormonal exposure. The addition of 10(-5) M L-NAME suppressed the antiaggregatory effect of 1 nM E2 or 10 nM Pg. Furthermore, we specifically quantified the NO generation by the 3H-citrulline technique. 10(-8) M E2 induced 2-fold increase of RAS citrulline production, while the increment induced by 10(-7) M Pg was 55% over control. Preincubation with 10(-5) M L-NAME completely suppressed the stimulatory action of 10(-9) M E2 or 10(-8) M Pg, confirming that the antiaggregatory factor released from the aortic tissue was NO. Preincubation with cycloheximide did not block the increment in NO induced by the hormones. In conclusion the present study provides for the first time evidence of acute, non-genomic effects of Pg on rat aorta NOS activity and platelet aggregation in coincidence with the results obtained with estradiol treatment.

Effect of aging on the mechanisms of PTH-induced calcium influx in rat intestinal cells.

We have investigated the effects of aging on parathyroid hormone (PTH) modulation of intracellular calcium homeostasis and their relationship to signal transduction pathways in isolated rat duodenal cells (enterocytes). PTH (10(-8)-10(-9) M) increased enterocyte (45)Ca(2+) influx and intracellular Ca(2+) concentration ([Ca(2+)](i)) to a greater extent (twofold and 50%, respectively) in aged (24 months) than in young (3 months) animals. The [Ca(2+)](i) response of old cells to the hormone was slower, lacking the early phase of changes in cytosolic Ca(2+). Ca(2+) influx induced by PTH was prevented by the protein kinase A antagonist Rp-cAMPS in both young and aged enterocytes, whereas neomycin and compound U73122, inhibitors of PLC-catalyzed phosphoinositide hydrolysis, abolished hormone-dependent Ca(2+) influx in young but had no effect on aged cells. Higher basal adenylyl cyclase (AC) activity and cAMP content were detected in old enterocytes. PTH increased the absolute levels of cAMP in aged cells and AC activity of microsomes isolated therefrom to a greater extent (>/= twofold) than in young enterocytes/membranes. In young cells, the hormone also induced a rapid and transient release of inositoltrisphosphate (IP(3)) and diacylglycerol (neomycin-sensitive) at 45 sec, and a delayed phase of DAG at 5 min (neomycin-insensitive). The early formation of IP(3) and DAG was blunted in aged animals. These results suggest that both the PLC and adenylyl cyclase cascades are involved in PTH stimulation of Ca(2+) influx in duodenal cells. During aging, however, only the cAMP pathway is operative, mediating a potentiation of the effects of the hormone. Additional studies are required to establish the relative role of PTH-dependent messenger systems in the regulation of intestinal calcium absorption and age-related abnormalities.

Acute modulation of Ca2+ influx on rat heart by 17beta-estradiol.

Estrogens initiate their action by binding to specific intracellular receptors and then acting on gene expression. In addition, there is growing evidence of a direct membrane effect via interaction with a cell surphase receptor. The aim of the present study was to investigate the acute effects of 17beta-estradiol on Ca2+ fluxes through second messenger pathways in rat cardiac muscle. Exposure of rat ventricle to low levels of 17beta-estradiol (10(-12)-10(-8) M) increased 45Ca2+ influx within 1 min (+38%); the response was biphasic, peaking at 2 and 5 min (+60 and +55%, respectively). The effect of the hormone on rat heart seems to be specific since 17alpha-estradiol, dihydrotestosterone, and progesterone were devoid of activity. The effect of 17beta-estradiol (5 min, 10(-10) M) was suppressed by nitrendipine (1 microM) and LaCl3 (10 microM), involving the activation of voltage-dependent Ca2+ channels in the acute increase of rat heart calcium influx by the hormone. 17Beta-estradiol rapidly increased cAMP content and PKA activity of rat cardiac muscle in parallel to the changes in Ca2+ uptake. In addition the cAMP antagonist Rp-cAMPS suppressed 17beta-estradiol-dependent Ca2+ influx. Altogether, the data suggest the involvement of the cAMP/PKA messenger system in the nongenomic modulation of Ca2+ influx in rat cardiac muscle by physiological levels of 17beta-estradiol.

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes.

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.

Parathyroid hormone stimulates calcium influx and the cAMP messenger system in rat enterocytes.

Direct effects of parathyroid hormone (PTH) on calcium uptake by isolated rat duodenal cell preparations enriched in enterocytes were investigated. PTH significantly stimulated enterocyte 45Ca2+ influx in a time-dependent (1-10 min) manner and at all doses tested (2 x 10(-13) to 10(-7) M). The Ca2+ channel antagonists verapamil (10 microM) and nitrendipine (1 microM) completely blocked the stimulation of Ca2+ influx by the hormone (10(-8) M). PTH markedly increased cAMP levels in rat duodenal cells (88, 167, and 67%, after 1, 2, and 3 min, respectively). In agreement with these observations, forskolin (adenylate cyclase activator), dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and Sp-cAMPS (cAMP analogs) mimicked, whereas Rp-cAMPS (cAMP antagonist) suppressed PTH and DBcAMP activation of enterocyte calcium uptake. Furthermore, the effects of DBcAMP were abolished by nitrendipine. These results show direct rapid effects of PTH on duodenal cells' Ca2+ influx, which involve the activation of a dihydropyridine-sensitive Ca2+ influx pathway and the cAMP second messenger system.

Effects of calcitriol and its analogues, calcipotriol (MC 903) and 20-epi-1alpha,25-dihydroxyvitamin D3 (MC 1288), on calcium influx and DNA synthesis in cultured muscle cells.

The fast actions of the secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3; calcitriol] and the synthetic analogues calcipotriol (MC 903) and 20-epi-1alpha,25(OH)2D3 (MC 1288) on cell calcium influx were compared in rat duodenum enterocytes as well as in cells from chick embryo skeletal muscle (myoblasts) and heart (myocytes), at various concentrations (10(-12) to 10(-8) M) and treatment intervals (1-10 min). In enterocytes, at a concentration of 10(-11) M, MC 1288 was significantly more active than 1,25(OH)2D3 in rapidly stimulating 45Ca2+ uptake by enterocytes (80 vs 38% above controls, respectively), whereas MC 903 was devoid of activity. However, calcipotriol increased Ca2+ influx in myocytes and myoblasts to a greater extent than the natural hormone, whereas MC 1288 was more active only in myoblasts. Analogously to 1,25(OH)2D3, the fast MC 903- and MC 1288-induced stimulation of 45Ca2+ uptake in enterocytes and muscle cells could be blocked by both verapamil and nifedipine. In addition, MC 903 and MC 1288 were more effective than 1,25(OH)2D3 in stimulating DNA synthesis in proliferating myoblasts and in inhibiting DNA synthesis in differentiating myoblasts. The results suggest, therefore, that modifications in the side-chain of the 1,25(OH)2D3 molecule increase its ability to modulate muscle cell Ca2+ metabolism and growth. These findings are potentially relevant for the development of analogues for the treatment of vitamin D-dependent myopathies.

Acute stimulation of intestinal cell calcium influx induced by 17 beta-estradiol via the cAMP messenger system.

Recent studies have provided evidence for nuclear estrogen receptor-mediated calcium transport in intestinal mucosal cells. The possibility that, in addition, estrogens directly stimulate intestinal Ca2+ fluxes through second-messenger pathways was investigated. Exposure of enterocytes isolated from female rat duodenum to low physiological levels of 17 beta-estradiol (10(-11), 10(-10) and 10(-8) M) rapidly (1-10 min) increased (50-170%) cell 45Ca2+ influx. 17 alpha-Estradiol, dihydrotestosterone and progesterone were devoid of activity, suggesting specificity of the estrogen effect. Maximum responses induced by 17 beta-estradiol (5 min at 10(-10) M) could be abolished to a great extent (84%) by pretreating the cells with verapamil (10 microM) and nitrendipine (1 microM), involving the activation of voltage-dependent Ca2+ channels in the fast increase of rat duodenal calcium uptake by the hormone. Evidence was obtained indicating that the acute estrogen stimulation of enterocyte Ca2+ influx is mediated by the cyclic AMP/PKA pathway. 17 beta-Estradiol rapidly increased cAMP content of rat duodenal cells in parallel to the changes in Ca2+ uptake. In addition, forskolin, dibutyryl cAMP and Sp-cAMPS mimicked and Rp-cAMPS suppressed the prompt 17 beta-estradiol-induced stimulation of Ca2+ influx. These results are consistent with a direct action of estrogens in the enterocyte, presumably a non-genomic one, initiated on the cell surface and resulting in rapid activation of the cAMP pathway and Ca2+ channels, which may be relevant for regulation of intestinal calcium transport.

Age-associated decrease in inositol 1,4,5-trisphosphate and diacylglycerol generation by 1,25(OH)2-vitamin D3 in rat intestine.

The hormonal form of vitamin D3, 1,25(OH)2-vitamin D3(1,25[OH]2D3), stimulates the breakdown of membrane phosphoinositides, generating inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in a variety of cell systems. Several studies suggest that alterations in the receptor-mediated phosphoinositide cascade are involved in the pathophysiology of aging. Therefore, the formation of IP3 and DAG were determined under basal conditions and after stimulation with physiological concentrations of 1,25(OH)2D3 in duodenum from young (3-mo-old) and aged (24-mo-old) rats. The hormone induced a transient and biphasic formation of IP3 and DAG. Values obtained in young rats peaking at 15 s (51% and 42% above basal levels for IP3 and DAG, respectively) and at 3 min (90% and 74% above basal levels for IP3 and DAG, respectively) were significantly decreased in duodenum from senescent animals (IP3: +20% and DAG: +18% above basal level at 15 s; and IP3: +18% and DAG: +29% above basal level at 3 min). The 1,25(OH)2D3-induced generation of DAG in both young and aged duodenum was effectively inhibited in the presence of neomycin, a phospholipase C (PLC) inhibitor, and was dependent on extracellular Ca2+. After the biphasic response, the levels of DAG generated by the hormone (10 min stimulation) remained elevated; the elevation occurred in the absence of IP3 production; and the elevated levels were not abolished by neomycin, implying that phospholipids other than phosphoinositides are the source of DAG. This 1,25(OH)2D3-dependent late phase of DAG generation was also diminished in aged animals. The precise molecular basis and the physiological significance of decreased liberation of IP3 and DAG by 1,25(OH)2D3 in the aged rat duodenum remains to be determined.

Rapid 1,25(OH)2-vitamin D3 stimulation of calcium uptake by rat intestinal cells involves a dihydropyridine-sensitive cAMP-dependent pathway.

The acute effects of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) on Ca2+ influx in isolated rat enterocytes were studied. The hormone significantly increased 45Ca2+ uptake by the cells within 1-10 min in a specific dose-dependent manner (10(-11)-10(-9) M) since 25(OH)D3 and 24,25(OH)2D3 were devoid of activity. The effects of 1,25(OH)2D3 were mimicked by the Ca2+ channel agonist BAY K8644 and completely abolished by nifedipine (1 microM) and verapamil (10 microM). Incubation of duodenal cells with 1,25(OH)2D3 rapidly (1-5 min) increased cAMP levels. Forskolin caused a rapid increase in Ca2+ uptake by enterocytes which was similar to the action of the hormone. Moreover, pretreatment of cells with the specific cAMP inhibitor Rp-cAMPS suppressed the changes in 45Ca influx induced by 1,25(OH)2D3. These results provide the first evidence involving Ca2+ channel activation through the cAMP pathway by 1,25(OH)2D3 in mammalian intestinal cells.

Regulation of Ca2+ uptake in skeletal muscle by 1,25-dihydroxyvitamin D3: role of phosphorylation and calmodulin.

Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in skeletal muscle. N-2'-o-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP), similarly as 1,25(OH)2D3 (5 x 10(-10) M), rapidly increased 45Ca uptake by soleus muscle from vitamin D-deficient chicks (+25% and +98% at 3 min and 10 min, respectively) in a dose-dependent manner. The effects of the cAMP analog (10 microM) and 1,25(OH)2D3 could be abolished by the Ca(2+)-channel blocker nifedipine and the calmodulin antagonist flufenazine. Calmodulin binding by two muscle microsomal proteins of 28 kDa and 30 kDa was stimulated within 1 min of exposure of the tissue to 1,25(OH)2D3. Direct effects of the sterol on membrane calmodulin binding were shown with isolated microsomes. The 1,25(OH)2D3-mediated rise of [125I]calmodulin binding to microsomal membranes was dependent on the presence of medium ATP. Forskolin (10 microM) and cAMP (10 microM) also increased [125I]calmodulin binding (+75% and +64%, respectively, with respect to controls). Pretreatment of microsomal membranes with cAMP-dependent protein kinase inhibitor (1 microgram/ml) or addition of alkaline phosphates (1 U/ml) after hormonal treatment caused complete inhibition of 1,25(OH)2D3-induced [125I]calmodulin binding to microsomal membrane proteins. These results imply modifications of membrane protein phosphorylation through the cAMP signal pathway and in turn of calmodulin binding in the mechanism by which 1,25(OH)2D3 rapidly stimulates skeletal muscle Ca2+ uptake.

Modulation of 1,25-dihydroxyvitamin D3-dependent Ca2+ uptake in skeletal muscle by protein kinase C.

In vitro studies have shown that short exposure (1-10 min) of vitamin D-deficient chick soleus muscle to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] causes an acute stimulation of tissue 45Ca uptake through voltage-gated Ca2+ channels, with parallel increases in cyclic AMP levels, adenylate cyclase activity and membrane protein phosphorylation. We further investigated the involvement of protein kinases in the rapid effects of 1,25(OH)2D3 on skeletal muscle. The hormone was found to stimulate the protein kinase C (PKC) activity of muscle membranes. The PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nM) was found to rapidly stimulate muscle 45Ca uptake, mimicking 1,25(OH)2D3. Increases of 68% and 46% were observed at 1 and 15 min of exposure to PMA respectively. The effects of PMA were dose-dependent (50-200 nM) and were specific, since the inactive analogue 4 alpha-phorbol was without effect. Analogously to the effects of the sterol, PMA-enhanced 45Ca uptake was abolished by the Ca2+ channel antagonists nifedipine (30 microM) and verapamil (50 microM). Staurosporine (10 nM), a PKC inhibitor, surprisingly potentiated 1,25(OH)2D3-dependent stimulation of 45Ca uptake. Exposure of skeletal muscle to PMA (100 nM) plus 1,25(OH)2D3 (1 nM) produced a less pronounced effect on 45Ca uptake than either agent alone. PMA also decreased muscle cyclic AMP levels. These results suggest a regulatory link between the two major transmembrane signalling systems in the mechanism of action of 1,25(OH)2D3 in skeletal muscle.

Cyclic AMP-dependent membrane protein phosphorylation and calmodulin binding are involved in the rapid stimulation of muscle calcium uptake by 1,25-dihydroxyvitamin D3.

Rapid in vivo effects of 1,25-dihydroxyvitamin D3 on muscle calcium metabolism have been reported. In vitro studies have shown that exposure of vitamin D-deficient chick soleus muscles to the sterol for 1-10 minutes causes a significant stimulation of tissue 45Ca uptake which can be suppressed by Ca channel blockers. A parallel increase in muscle membrane calmodulin content that could be mimicked by forskolin was observed. Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3. Like the sterol, forskolin (10 microns) rapidly increased (+48% at 5 min) soleus muscle 45Ca uptake and its effect could be reversed by nifedipine (50 microns). In agreement with these observations, 1,25-dihydroxyvitamin D3 markedly elevated tissue cAMP levels within 45 seconds to 5 minutes of treatment in a dose-dependent manner (10(-11)-10(-7) M). Moreover, incubation of isolated muscle microsomes with 1,25-dihydroxyvitamin D3 increased adenylate cyclase activity and caused a similar profile of stimulation of protein phosphorylation with [gamma-32P]-ATP as forskolin. Major changes were detected in proteins whose calmodulin binding ability has been previously shown to be increased by 1,25-dihydroxyvitamin D3. In addition, the calmodulin antagonists fluphenazine and compound 48/80 abolished the increase in muscle Ca uptake and membrane calmodulin content produced by the sterol. The results suggest that 1,25-dihydroxyvitamin D3 activates muscle Ca channels through a direct membrane action which involves cAMP-dependent protein phosphorylation and calmodulin binding.

Stimulation of calmodulin binding to skeletal muscle membrane proteins by 1,25-dihydroxy-vitamin D3.

Previous work has shown that 1,25-dihydroxy-vitamin D3 rapidly increases calmodulin levels of skeletal muscle membranes without altering the muscle cell calmodulin content. Therefore, the effects of the sterol on the binding of calmodulin to specific muscle membrane proteins were investigated. Soleus muscles from vitamin D-deficient chicks were treated in vitro for short intervals (5-15 min) with physiological concentrations of 1,25-dihydroxy-vitamin D3. Proteins of mitochondria and microsomes isolated by differential centrifugation were separated on sodium dodecyl sulfate polyacrylamide gels. Calmodulin-binding proteins were identified by a [125I]calmodulin gel overlay procedure followed by autoradiography. 1,25-Dihydroxy-vitamin D3 increased the binding of labelled calmodulin to a major, calcium-independent, calmodulin-binding protein of 28 Kda localized in microsomes, and to minor calmodulin-binding proteins of 78 and 130 Kda proteins localized in mitochondria. The binding of [125I]calmodulin to these proteins was abolished by flufenazine or excess non-radioactive calmodulin. 1,25-Dihydroxy-vitamin D3 rapidly increased muscle tissue Ca uptake and cyclic AMP levels and stimulated the phosphorylation of several membrane proteins including those whose calmodulin-binding capacity potentiates. Analogously to the sterol, forskolin increased membrane calmodulin content, calmodulin binding to the 28 Kda microsomal protein and 45Ca uptake by soleus muscle preparations. Forskolin also induced a similar profile of changes in muscle membrane protein phosphorylation as the hormone. These results suggest that 1,25-dihydroxy-vitamin D3 affects calmodulin distribution in muscle cells through cyclic AMP-dependent phosphorylation of membrane calmodulin-binding proteins. These changes may play a role in the stimulation of muscle Ca uptake by the sterol.

1,25 Dihydroxyvitamin D3 affects calmodulin distribution among subcellular fractions of skeletal muscle.

1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.