Prolactin directly stimulated the solvent drag-induced calcium transport in the duodenum of female rats

Prolactin has been reported to stimulate the calcium absorption of the duodenum where three components of the active calcium transport, namely transcellular active, voltage-dependent and solvent drag-induced calcium transport, were identified. It was known that the transcellular active, but not the voltage-dependent, duodenal calcium transport was directly stimulated by prolactin. The present study thus aimed to evaluate the direct action of prolactin on the solvent drag-induced duodenal calcium transport by using the Ussing chamber technique. The jejunum was used as a reference for the existence of solvent drag and the widening of tight junction induced by cytochalasin E. Results showed that the solvent drag-induced calcium transport existed in both intestinal segments, but the magnitude was significantly greater in the duodenum (29.27+/-2.27 vs. 17.31+/-1.65 nmol h(-1) cm(-2), P<0.001). We further demonstrated that 200, 600 and 800, but not 1000 ng/ml, prolactin significantly promoted the solvent drag-induced duodenal calcium transport in a dose-response manner, i.e. from the control value of (nmol h(-1) cm(-2)) 24.31+/-2.36 to 45.42+/-3.47 (P<0.01), 63.82+/-5.28 (P<0.001) and 53.93+/-5.41 (P<0.01), respectively. However, prolactin did not manifest any effect on the jejunum. Because the paracellular transport was suggested to be size-selective as well as charge-selective, further experiments were designed to evaluate the mechanism by which prolactin stimulated the solvent drag-induced calcium transport. The duodenum was exposed to 20 microM cytochalasin E, 600 ng/ml prolactin or the combination of both in the presence of a paracellular marker 3H-mannitol, while the jejunum was a positive reference. The results showed that, in the jejunum, cytochalasin E alone and cytochalasin E plus prolactin significantly increased the mannitol fluxes from (micromol h(-1) cm(-2)) 0.29+/-0.04 to 0.49+/-0.03 (P<0.05) and 0.48+/-0.05 (P<0.05), respectively, while having no effect on the calcium fluxes. Prolactin alone had no effect on the jejunal calcium flux. In the duodenum, neither mannitol nor calcium fluxes were enhanced by cytochalasin E, however, prolactin still increased the solvent drag-induced calcium flux from 27.74+/-2.41 to 51.03+/-4.35 nmol h(-1) cm(-2) (P<0.001). It was concluded that prolactin directly stimulated the solvent drag-induced duodenal calcium transport in a dose-response and biphasic manner without the widening of tight junction.     

Long-term prolactin exposure differentially stimulated the transcellular and solvent drag-induced calcium transport in the duodenum of ovariectomized rats

Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P < 0.001) and 9.78 +/- 0.72 to 15.99 +/- 1.75 (P < 0.001) nmol.hr(-1).cm(-2), respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 +/- 10.12 to 47.77 +/- 5.42 nmol.hr(-1).cm(-2) (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.     

Prolactin directly stimulates transcellular active calcium transport in the duodenum of female rats

Prolactin has been postulated to be a novel calcium-regulating hormone during pregnancy and lactation. It stimulates both passive and active duodenal calcium transport in several experimental models. Our study was performed on sexually mature female Wistar rats (200-250 g) to study the direct action of prolactin on calcium transport in the duodenum using the Ussing chamber technique. To evaluate the effect of prolactin on total calcium transport in the duodenum, we intraperitoneally injected rats with 0.4, 0.6, and 0.8 mg/kg prolactin. The total calcium transport was divided into voltage-dependent, solvent drag-induced, and transcellular active fluxes by applying short-circuit current and by mucosal glucose replacement with mannitol. The effect of prolactin on each flux was studied separately. Finally, to evaluate the direct action of prolactin on duodenal transcellular active flux, we directly exposed duodenal segments to prolactin that had been added to the serosal solution with or without calcium transport inhibitors. We found that 0.6 and 0.8 mg/kg prolactin ip significantly increased the total mucosa-to-serosa calcium flux from the control value (nmol x hr(-1) x cm(-2)) of 34.53+/-6.81 to 68.07+/-13.53 (P < 0.05) and 84.43+/-19.72 (P < 0.01), respectively. Prolactin also enhanced the solvent drag-induced calcium flux and transcellular active calcium flux, but not the voltage-dependent calcium flux. The duodenal segments directly exposed to 200, 400, and 800 ng/mL prolactin showed a significant increase in the transcellular active calcium absorption in a dose-dependent manner, i.e., from the control value (nmol x hr(-1) x cm(-2)) of 2.94+/-0.47 to 5.45+/-0.97 (P < 0.01), 8.09+/-0.52 (P < 0.001), and 18.42+/-2.92 (P < 0.001), respectively. Its direct action was inhibited by mucosal exposure to 50 microM lanthanum chloride, a calcium transporter protein competitor, and serosal exposure to 0.1 mM trifluoperazine, a Ca2+-ATPase inhibitor. These studies demonstrate that the duodenum is a target organ of prolactin, which enhances transcellular active calcium transport.     

Chronic metabolic acidosis upregulated claudin mRNA expression in the duodenal enterocytes of female rats

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague-Dawley rats given 1.5% NH(4)Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.     

Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats

Elevated plasma levels of prolactin (PRL) have been reported in several physiological and pathological conditions, such as lactation, prolactinoma, and dopaminergic antipsychotic drug uses. Although PRL is a calcium-regulating hormone that stimulates intestinal calcium absorption in lactating rats, whether PRL is capable of stimulating calcium absorption in male rats has been elusive. Herein, the transepithelial calcium transport and electrical characteristics were determined in ex vivo duodenal tissues of male rats by Ussing chamber technique. We found that PRL receptors were abundantly present in the basolateral membrane of the duodenal epithelial cells. PRL (200–800 ng/mL) markedly increased the active duodenal calcium transport in a dose-dependent fashion without effect on the transepithelial resistance. The PRL-enhanced active duodenal calcium transport was completely abolished by L-type calcium channel blocker (nifedipine) as well as inhibitors of the major basolateral calcium transporters, namely plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger. Several intracellular mediators, such as JAK2, MEK, PI3K and Src kinase, were involved in the PRL-enhanced transcellular calcium transport. Moreover, PRL also stimulated the paracellular calcium transport in the duodenum of male rats in a PI3K-dependent manner. In conclusion, PRL appeared to be a calcium-regulating hormone in male rats by enhancing the L-type calcium channel-mediated transcellular and the paracellular passive duodenal calcium transport. This phenomenon could help restrict or alleviate negative calcium balance and osteoporosis that often accompany hyperprolactinemia in male patients.     

Functional organization of calcium stores in polarized secretory cells and transcellular calcium transport

In this short review, we will first discuss localized cytoplasmic calcium signals in pancreatic acinar cells. In the second part of the review, we will describe recently discovered polarized calcium efflux and calcium propagation through the lumen of the endoplasmic reticulum — ER (a phenomenon we have termed “calcium tunnelling”). Finally, we will present a hypothesis concerning the roles that these mechanisms could play in transcellular calcium flux.     

The pyroantimonate reaction and transcellular transport of calcium in rat molar enamel organs

The distribution of calcium in the cells of the enamel organ of developing rat molar tooth germs was studied by the pyroantimonate method. It was found that there was a specific localization to the inner leaflet of the plasma membrane of both secretory and maturation phase ameloblasts. This information can be used to support the model for transcellular transport of calcium involving membrane fluidity, with phosphatidylserine as a carrier (Reith 1983). It can also support an alternative model involving movement of calcium ions over a surface of acidic phospholipids on the inner leaflet of the plasma membrane, without involving the necessity for membrane fluidity.     

The pyroantimonate reaction and transcellular transport of calcium in rat molar enamel organs

Summary The distribution of calcium in the cells of the enamel organ of developing rat molar tooth germs was studied by the pyroantimonate method. It was found that there was a specific localization to the inner leaflet of the plasma membrane of both secretory and maturation phase ameloblasts. This information can be used to support the model for transcellular transport of calcium involving membrane fluidity, with phosphatidylserine as a carrier (Reith 1983). It can also support an alternative model involving movement of calcium ions over a surface of acidic phospholipids on the inner leaflet of the plasma membrane, without involving the necessity for membrane fluidity.Deceased 9 May 1985     

Modeling of transcellular Ca transport in rat duodenum points to coexistence of two mechanisms of apical entry

Employing realistic parameters, we have demonstrated that a relatively simple mathematical model can reproduce key features of steady-state Ca2+ transport with the assumption of two mechanisms of Ca2+ entry: a channel-like flux and a carrier-mediated transport. At low luminal [Ca2+] (1-5 mM), facilitated entry dominates and saturates with Km = 0.4 mM. At luminal [Ca2+] of tens of millimolar, apical permeability is dominated by the channel flux that in turn is regulated by cytosolic Ca2+. The model reproduces the linear relationship between maximum Ca2+ transport rate and intestinal calbindin D9K (CaBP) content. At luminal [Ca2+] > 50 mM, local sensitivity analysis shows transcellular transport to be most sensitive to variations in CaBP. At low luminal [Ca2+], transport becomes sensitive to apical entry regulation. The simulations have been run within the Virtual Cell modeling environment, yielding the time course of external Ca2+ and spatiotemporal distributions of both intracellular Ca2+ and CaBP. Coexistence of two apical entry mechanisms accords with the properties of the duodenal Ca2+ transport protein CaT1 and the epithelial Ca2+ channel ECaC.     

Acute metabolic acidosis inhibits muscle protein synthesis in rats

In this study, we investigated the effect of acute metabolic acidosis on tissue protein synthesis. Groups of rats were made acidotic with intragastric administration of NH(4)Cl (20 mmol/kg body wt every 12 h for 24 h) or given equimolar amounts of NaCl (controls). Protein synthesis in skeletal muscle and a variety of different tissues, including lymphocytes, was measured after 24 h by injection of l-[(2)H(5)]phenylalanine (150 micromol/100 g body wt, 40 moles percent). Results show that acute acidosis inhibits protein synthesis in skeletal muscle (-29% in gastrocnemius, -23% in plantaris, and -17% in soleus muscles, P < 0.01) but does not affect protein synthesis in heart, liver, gut, kidney, and spleen. Protein synthesis in lymphocytes is also reduced by acidosis (-8%, P < 0.05). In a separate experiment, protein synthesis was also measured in acidotic and control rats by a constant infusion of l-[(2)H(5)]phenylalanine (1 micromol.100 g body wt(-1).h(-1)). The results confirm the earlier findings showing an inhibition of protein synthesis in gastrocnemius (-28%, P < 0.01) and plantaris (-19%, P < 0.01) muscles but no effect on heart and liver by acidosis. Similar results were also observed using a different model of acute metabolic acidosis, in which rats were given a cation exchange resin in the H(+) (acidotic) or the Na(+) (controls) form. In conclusion, this study demonstrates that acute metabolic acidosis for 24 h depresses protein synthesis in skeletal muscle and lymphocytes but does not alter protein synthesis in visceral tissues. Inhibition of muscle protein synthesis might be another mechanism contributing to the loss of muscle tissue observed in acidosis.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

枸橼酸转运蛋白mRNA在代谢性酸中毒大鼠肾组织的表达

文章报道了代谢性酸中毒时大鼠肾组织两种钠离子依赖的枸橼酸膜转运蛋白ｍＲＮＡ表达量的变化。给雌性Ｗｉｓｔａｒ大鼠喂含０．２８ｍｏｌ／Ｌ ＮＨ４Ｃｌ饮用水诱导产生代谢性酸中毒。喂酸后分别于１、３、７ｄ处死大鼠,测定血浆ＨＣＯ＾－３浓度的变化。以Ｎｏｒｔｈｅｒｎ杂交方法,用钠离子依赖的枸橼酸膜转运蛋白１（ＳＤＣＴ１）探针及钠离子依赖的枸橼酸膜转运蛋白２（ＳＤＣＴ２）探针,分别检测肾皮质枸橼酸转运蛋白     

Regulation of duodenal insulin-like growth factor I and active calcium transport by ovariectomy in female rats.

There is a significant body of data that supports the concept that reproductive hormones in females have effects on duodenal calcium transport that are not mediated via altered circulating concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Previously, we have shown parallel alterations in duodenal Ca transport and longitudinal bone growth rate in sexually maturing female rats in response to ovariectomy and estradiol (E) treatment of ovariectomized (OVX) rats (OVX+E) without any change in circulating levels of 1,25(OH)2D or parathyroid hormone. Results are presented here from experiments designed to: (i) further explore the relationship between 1,25(OH)2D and ovarian status in the regulation of duodenal calcium transport, and (ii) determine whether OVX and E replacement alter circulating and duodenal levels of insulin-like growth factor I (IGF-I) that might be related to effects on Ca transport. Growth hormone, which has been shown to affect intestinal Ca absorption and vitamin D metabolism, is thought to act indirectly by stimulating IGF-I. Six-week-old female rats were OVX, given estradiol implants (OVX+E), and fed a diet containing either 0.5% or 0.1% Ca for 3 weeks. In both diet groups, the OVX animals exhibited a higher level of Ca transport, as measured by the everted gut sac method, than either the intact controls or the OVX+E group; there was no difference in calcium transport between the different diet groups. Although there was no difference in circulating levels of 1,25(OH)2D among the intact, OVX, and OVX+E groups fed either diet, animals fed the 0.1% Ca diet had higher circulating levels of 1,25(OH)2D than those fed the 0.5% Ca diet. There was no difference in duodenal levels of calbindin9K among intact, OVX, and OVX+E animals in either diet group, although the animals fed the 0.1% Ca diet had higher levels of calbindin9K than the animals fed the 0.5% Ca diet. In animals fed the 0.5% Ca diet, OVX resulted in elevated serum and duodenal levels of IGF-1, as compared with intact and OVX+E animals on the same diet. In animals fed the 0.1% Ca diet, there was no elevation of IGF-I in the OVX group relative to intact and OVX+E animals. These results lend additional support to the concept that alterations in duodenal active calcium transport that occur with alterations in ovarian hormones are not mediated by changes in serum levels of 1,25(OH)2D, but may be related to some factor related to growth, possibly IGF-I.     

Phosphate transport in the duodenum and jejunum of goats and its adaptation by dietary phosphate and calcium

Endogenous P(i) recycling is a characteristic feature of the P homeostasis in ruminants. A pronounced salivary P(i) secretion into the rumen is balanced by a high intestinal P(i) absorption and an almost complete renal P(i) reabsorption. In monogastric animals, the major P(i) transport mechanism across the apical membrane of the enterocyte is an Na(+)-dependent transport mediated by NaPi cotransporter type IIb. In ruminants, an Na(+)-, as well as an H(+)-dependent, P(i) transport system seems to exist in the small intestines. Therefore, morphological localization, type of ionic dependence, and ability to adapt to dietary P or Ca restriction of duodenal and jejunal P(i) transport were characterized in goats. In the duodenum, there was an H(+)-dependent, Na(+)-sensitive P(i) transport system that did not belong to the NaPi type II family and was not influenced by dietary P or Ca restriction. In contrast, in the jejunum, there was an Na(+)-dependent, H(+)-sensitive P(i) transport mainly mediated by NaPi IIb. P restriction stimulated the NaPi IIb protein expression, resulting in higher P(i) transport capacity.     

Role of acidosis-induced increases in calcium on PTH secretion in acute metabolic and respiratory acidosis in the dog

Recently, we showed that both acute metabolic acidosis and respiratory acidosis stimulate parathyroid hormone (PTH) secretion in the dog. To evaluate the specific effect of acidosis, ionized calcium (iCa) was clamped at a normal value. Because iCa values normally increase during acute acidosis, we now have studied the PTH response to acute metabolic and respiratory acidosis in dogs in which the iCa concentration was allowed to increase (nonclamped) compared with dogs with a normal iCa concentration (clamped). Five groups of dogs were studied: control, metabolic (clamped and nonclamped), and respiratory (clamped and nonclamped) acidosis. Metabolic (HCl infusion) and respiratory (hypoventilation) acidosis was progressively induced during 60 min. In the two clamped groups, iCa was maintained at a normal value with an EDTA infusion. Both metabolic and respiratory acidosis increased (P < 0.05) iCa values in nonclamped groups. In metabolic acidosis, the increase in iCa was progressive and greater (P < 0.05) than in respiratory acidosis, in which iCa increased by 0.04 mM and then remained constant despite further pH reductions. The increase in PTH values was greater (P < 0.05) in clamped than in nonclamped groups (metabolic and respiratory acidosis). In the nonclamped metabolic acidosis group, PTH values first increased and then decreased from peak values when iCa increased by > 0.1 mM. In the nonclamped respiratory acidosis group, PTH values exceeded (P < 0.05) baseline values only after iCa values stopped increasing at a pH of 7.30. For the same increase in iCa in the nonclamped groups, PTH values increased more in metabolic acidosis. In conclusion, 1) both metabolic acidosis and respiratory acidosis stimulate PTH secretion; 2) the physiological increase in the iCa concentration during the induction of metabolic and respiratory acidosis reduces the magnitude of the PTH increase; 3) in metabolic acidosis, the increase in the iCa concentration can be of sufficient magnitude to reverse the increase in PTH values; and 4) for the same degree of acidosis-induced hypercalcemia, the increase in PTH values is greater in metabolic than in respiratory acidosis.