Intestinal Na(+)/Ca(2+) exchanger protein and gene expression are regulated by 1,25(OH)(2)D(3) in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Intestinal Na/Ca exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

Vitamin D3 and 1,25-dihydroxyvitamin D3 stimulate the skeletal muscle-calcium mobilization in rachitic chicks

The Ca content in skeletal muscle relative to vitamin D3 intake was studied in chicks. It was found that the Ca content in rachitic chick muscle was significantly higher than normal and it decreased with vitamin D3 treatment. In 4-week-old chicks fed a vitamin D-deficient diet, the Ca content in leg muscle reached 9.86 +/- 1.07 mg/100 g wet wt, although in chicks receiving vitamin D3 in doses of 100 and 500 IU/kg diet, it was 7.80 +/- 0.72 and 6.08 +/- 0.61 mg/100 g wet wt, respectively. A single i.m. dose of 0.50 micrograms of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or vitamin D3 caused a dramatic decrease in the muscle Ca content by 3 to 6 h after the injection. A simultaneous rise in the Ca level in blood serum was observed. However, at this time the Ca binding protein content in duodenal mucosa and the stimulation of Ca absorption were negligible. These findings allow the conclusion that the vitamin D deficiency in chicks leads to a surplus Ca accumulation in skeletal muscle. The administration of vitamin D3 or its metabolites causes rapid Ca release during the first 6 h. This may be the source of the Ca level increase in blood serum. In this respect 1,25(OH)2D3 was much more effective than vitamin D3.     

Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption

A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)₂D₃, to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and⁴⁷Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope,⁴⁴Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the⁴⁴Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)₂D₃ was an increased tissue uptake of luminal⁴⁷Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2–4 h after the 1,25(OH)₂)D₃ dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8–16 h,⁴⁷Ca absorption was maximal and⁴⁴Ca was sparsely distributed in the intestinal tissue.⁴⁷Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue⁴⁴Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)₂D₃ on the intestinal Ca absorption     

Hormonal effects on the sulfhydryl groups associated with intestinal brush border membrane proteins.

Previous studies demonstrated that the administration of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) to cholecalciferol-deficient chicks rapidly increases the reactivity and amount of the sulfhydryl (HS-) groups in intestinal brush border membranes (BBM). In the present study, the tissue and hormonal specificity of this effect was investigated. The HS- groups of intestinal and renal BBM were enhanced by vitamin D-3 and/or 1,25(OH)2D3, but no change was noted in isolated intestinal mitochondria and purified intestinal basolateral membranes, cardiac sarcolemma and erythrocyte membranes. Other steroid hormones including estradiol, testosterone, aldosterone, cortisol, dexamethasone and progesterone, yielded a response similar to 1,25(OH)2D3 on BBM HS- groups. Triiodothyronine and retinoic acid also resulted in an increase in intestinal BBM HS- groups. In a kinetic approach, using a specific sulfhydryl fluorescent probe (N-7-dimethylamino-4-coumarin-3-yl-maleimide, DACM), the reactivity of the BBM HS- groups was increased by estrogen and testosterone, as was previously shown for 1,25(OH)2D3. Intestinal BBM proteins, labeled with DACM, were separated by gel electrophoresis. Fluorescence scans of the gel showed two heavily labeled bands, one of 110 kDa, putatively brush border myosin I, and one of 43 kDa, putatively actin. Labeling of the 110 kDa protein was increased by 1,25(OH)2D3 and estradiol. Further studies are required to elucidate the physiological meaning of these hormone-mediated increases in reactivity and amount of the BBM sulfhydryl groups, as well as the nature of the intermediate biochemical reactions involved in this response.     

Regulation of kidney calcium-binding protein in the bird (Gallus domesticus)

The possible involvement of plasma calcium and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the regulation of the concentration of kidney calcium-binding protein (CaBP) was investigated. Chicks were fed diets varying in Ca2+ and P, with or without vitamin D. CaBP and 1,25(OH)2D3 were determined by competitive binding assays. A significant correlation between plasma and kidney 1,25(OH)2D3 was found, the linear regression equation of best-fit was plasma 1,25(OH)2D3 = 0.14 + 1.56 kidney 1,25(OH)2D3. In the vitamin D-fed chicks, kidney CaBP varied independently of the circulating or organ level of 1,25(OH)2D3 (P greater than 0.05), but was lower in the vitamin D-deficient than in the vitamin D-fed birds. A significant correlation was observed between kidney CaBP and plasma calcium (Cap). The regression equations were CaBP = Cap/(85.57-4.00 Cap) (R = 0.845) and CaBP = 0.0558 + 0.0404 Cap (R = 0.749), for vitamin D-treated and vitamin D-deficient chicks, respectively. The results suggest that the concentration of kidney CaBP is modulated by plasma calcium, but one or more of the vitamin D metabolites may be required for its synthesis.     

Human colon cell line HT-29: characterisation of 1,25-dihydroxyvitamin D3 receptor and induction of differentiation by the hormone

The human colon carcinoma cell line HT-29 differentiates into functional enterocytes upon replacement of glucose by galactose in the culture medium. Since the differentiation of other types of cells is associated with the modulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) receptor concentrations and since enterocytes are classical target cells for 1,25(OH)2D3 we have examined the HT-29 cells to determine whether the differentiated and undifferentiated stages could be directly linked to the presence of 1,25(OH)2D3 receptors. HT-29 cells were grown in Dulbecco's modified medium containing 10% fetal calf serum (FCS) and glucose or galactose. Cell differentiation was assessed by measuring the brush border hydrolase, maltase. 1,25(OH)2D3 receptors were studied in the cells after 48 h without FCS. Nuclear uptake was measured in intact dispersed cells and the receptor protein was further characterized by vitamin D metabolite binding specificity, sucrose density gradient analysis and binding to DNA-cellulose. Maltase activity was 5-fold greater in differentiated HT-29 cells than in undifferentiated cells. Scatchard analysis showed a highly specific saturable (9500 sites per cell) high affinity (2 x 10(-10) M), binding of 1,25(OH)2D3 in undifferentiated cells. This receptor-like protein sedimented at 3.3S, bound to and eluted from DNA-cellulose and had all the characteristics of a 1,25(OH)2D3 receptor. No specific binding was detected in differentiated HT-29 cells. The presence of 1,25(OH)2D3 receptors in undifferentiated HT-29 cells implies that these cells are targets for vitamin D. The maltase activity increased significantly when undifferentiated cells were exposed to 1,25(OH)2D3 for 5-6 days, indicating that the hormone can promote differentiation of HT-29 cells. These results demonstrate that HT-29 cells can provide a new model for studying steroid receptor regulation and cell differentiation.     

The effect of essential fatty acid deficiency on the stimulation of intestinal calcium transport by 1,25-dihydroxyvitamin D3

The effect of altering the lipid composition of the brush-border membrane on the ability of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to stimulate calcium transport across the intestinal mucosa was examined by raising chicks on a vitamin D, essential fatty acid-deficient diet (-DEFAD) and measuring calcium absorption from duodenal sacs in situ and calcium uptake into brush-border membrane vesicles in vitro. Administration of 1,25-(OH)2D3 to -DEFAD and to -D control chicks led to the same increase in calcium transport in situ, whereas calcium transport in isolated brush-border membrane vesicles was not stimulated in the EFAD group, but responded normally in the control group. When the incubation temperature was increased to 34 degrees C, brush-border membrane vesicles from 1,25-(OH)2D3-treated essential fatty acid-deficient (+DE-FAD) chicks accumulated calcium at a faster rate than did vesicles from -DEFAD chicks. There was a marked decrease in the linoleic acid content and an increase in the oleic acid content of both the total lipid extract of the brush-border membrane as well as the phosphatidylcholine and phosphatidylethanolamine fractions, which could explain the temperature sensitivity of the in vitro system. When the diet of the EFAD chicks was supplemented with linoleic acid, the rate of calcium uptake into subsequently isolated vesicles from +DE-FAD chicks correlated with the amount of linoleic acid in the brush-border membranes. These results support the concept that the action of 1,25-(OH)2D3 on membrane lipid turnover and structure plays a critically important role in the 1,25-(OH)2D3-mediated cellular transport responses.     

Binding properties of duodenal 1,25-dihydroxyvitamin D3 receptors as affected by phosphorus depletion in lactating goats

1. Capacity and affinity of duodenal 1,25(OH)2D3 receptors were measured in P depleted goats and in control animals kept on an adequate P supply. Plasma concentrations of Pi, Ca and vitamin D3 metabolites and activity of plasma alkaline phosphatase were measured to characterize the effects of P depletion. 2. During P depletion plasma Pi concentrations decreased significantly whereas plasma Ca and alkaline phosphatase activity increased. No changes were recorded for plasma vitamin D3, 25OHD3 and 1,25(OH)2D3 concentrations. 3. P depletion resulted in a significant decrease of the equilibrium dissociation constant Kd of duodenal 1,25(OH)2D3 receptors without affecting the maximum binding capacity.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

Isolation and identification of 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3. New metabolites of vitamin D3 produced by a C-24 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.     

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).     

Ca2(+)-channel agonist BAY K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2+ transport in chick perfused duodenum.

To further understand the molecular mechanism by which 1,25(OH)2-vitamin D3 [1,25(OH2D3] rapidly stimulates intestinal calcium transport (termed "transcaltachia"), the effect of the calcium channel agonist BAY K8644 was studied in vascularly perfused duodenal loops from normal, vitamin D-replete chicks. BAY K8644, 2 mu M, was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25(OH)2D3. The sterol and the Ca2+ channel agonist both increased 45Ca2+ transport 70% above control values within 2 min and 200% after 30 min of vascular perfusion. The effect of the Ca2+ channel agonist was dose dependent. Also, 1,25(OH)2D3-enhanced transcaltachia was abolished by the calcium channel blocker nifedipine. Collectively, these results suggest the involvement of 1,25(OH)2D3 in the activation of basal lateral membrane Ca2+ channels as an early effect in the transcaltachic response.