Curtis S. Fullmer

Intestinal calcium transport and calcium extrusion processes at the basolateral membrane

The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.

Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography☆

A method is described for the facile identification of half-cystine residues in peptides from protein digests. Combined use of S-beta-(4-pyridylethylation) of cysteine residues with 4-vinyl pyridine and peptide mapping by reversed-phase high-performance liquid chromatography provides the basis for this procedure. Essentially simultaneous, in-line effluent monitoring at 220, 280, and 254 nm provides highly sensitive, specific, and instantaneous identification of all peptide peaks, as well as those containing tryptophan and half-cystine residues, respectively. The resultant cysteine-derivatized peptides are well suited to amino acid compositional and protein sequence analysis, and are protected from loss during enzymatic digestion and/or peptide mapping.

Embryonic chick intestine in organ culture: stimulation of calcium transport by exogenous vitamin D-induced calcium-binding protein.

Abstract Vitamin D 3 or a potent metabolite, 1,25-dihydroxycholecalciferol, induces calcium-binding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen

SummaryThe localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.

Intestinal calcium and lead absorption : effects of dietary lead and calcium

The combined effects of dietary calcium (Ca) and lead (Pb) status on intestinal Ca and Pb absorption and related parameters were investigated in young growing chicks. Dietary Pb intake resulted in two remarkable, apparently independent and essentially opposite effects on intestinal Ca and Pb absorption, depending on dietary Ca and Pb levels and duration of treatment. The initial response (1 week) to Ca deficiency was stimulated Ca absorption and calbindin-D level, regardless of dietary Pb intake. The later response (2 weeks) was a reversal, by Pb, of the early phase stimulation. Intestinal Pb absorption was similarly enhanced by Ca deficiency initially, and this response was also inhibited by prolonged dietary Pb intake. Ingestion of Pb by chicks fed adequate Ca resulted in generally elevated intestinal Ca absorption and calbindin-D levels after both 1 and 2 weeks. Intestinal Pb absorption was also increased in the adequate Ca situation, but only after 2 weeks at the lower levels of dietary Pb. The results underscore the complicated nature of Pb-Ca interactions and demonstrate the importance of thorough characterization of the animal model system.

Modulation of chick intestinal and renal calbindin gene expression by dietary vitamin D3, 1,25-dihydroxyvitamin D3, calcium and phosphorus

Synthetic oligonucleotide probes complementary to chick calbindin-28 kDa-mRNA were used to study the latters regulation and relationship to calbindin in the chick. The effects of vitamin D3 sources and dietary alteration on the genomic expression were characterized by Northern blot and solution hybridization. Intestinal calbindin and its mRNA were almost absent in vitamin D-deficient chicks and were not affected by dietary alteration. Renal calbindin and its mRNA were lower in the vitamin D-deficient than in vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-fed chicks. In the same animal, renal calbindin mRNA and calbindin were higher than intestinal. In vitamin D3-fed chicks, dietary calcium (Ca) or phosphorus (P) restriction induced, and high dietary Ca inhibited, intestinal calbindin and its mRNA synthesis. In the same chicks, dietary P restriction induced renal calbindin mRNA and calbindin synthesis. In 1,25-(OH)2D3-fed chicks, dietary P restriction induced and high dietary Ca inhibited the synthesis of intestinal and renal calbindin. The results suggest that: (a) most of the changes in renal and intestinal calbindin could be attributed to the changes in the mRNA; (b) the adaptation to dietary Ca and P alterations requires vitamin D metabolites; (c) high dietary Ca affects intestinal and renal calbindin-mRNA and calbindin via mechanisms independent of kidney 1-hydroxylase; and (d) plasma Ca and renal calbindin or its mRNA tend to change together in vitamin D-deficient or vitamin D3-fed, but not in 1,25(OH)2D3-fed chicks.

Intestinal calcium absorption: calcium entry.

Any consideration of calcium entry into the cell must recognize that it is the initial event in a complex sequentially integrated process. Any step in this process, when viewed individually and in isolation, may appear to be of overwhelming importance, but this need not be an accurate reflection of its relative role in the overall process. Calcium entry may be of substantial importance in terms of calcium transport rate or capacity under certain circumstances, but it is most likely not the sole limiting step in calcium absorption. The Symposium papers that follow stress the importance of additional factors and events that have been implicated in intestinal calcium absorption.

Effect of dietary calcium and lead status on intestinal calcium absorption

Dietary lead intake was demonstrated to result in two very different effects on intestinal calcium absorption and associated parameters, depending on dietary calcium status. Normal growing 14-day-old chicks were fed diets either low (0.05%) or adequate (1.2%) in calcium and containing varying levels of lead (0%-0.8%) for an additional 10 days. In chicks fed the low calcium diet, ingested lead inhibited intestinal 47Ca absorption, and intestinal calbindin D and alkaline phosphatase synthesis in a dose-dependent fashion. Even at the highest levels of lead, however, this inhibition was limited to the stimulation of these parameters by low dietary calcium. In chicks fed the normal calcium diet, lead exposure did not diminish intestinal 47Ca absorption, or calbindin D or alkaline phosphatase synthesis, but actually elevated the values of these parameters at the higher lead exposure levels. The results indicate that the primary effect of lead, in both cases, occurs at or prior to intestinal protein synthesis and most likely involves the cholecalciferol endocrine system rather than any direct interactions between lead and calcium at the intestinal level.

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

Effect of cadmium administration on intestinal calcium absorption and vitamin D-dependent calcium-binding protein

The effects of cadmium on intestinal calcium absorption and calcium-binding protein (CaBP) were investigated in chicks by means of the in situ ligated duodenal loop technique. Dietary cadmium, administered in the feed or by gastric intubation, resulted in significant declines in intestinal calcium absorption and mucosal calcium-binding protein concentrations. Cadmium chloride injected directly into the ligated loop of naive chicks also diminished calcium absorption and CaBP concentrations in an apparently dose-response related fashion. No adverse effects of cadmium administration on either the 25- or 1α-hydroxylation reactions of vitamin D were observed. While the general effect of cadmium administration was a reduction in intestinal calcium absorption, plasma calcium levels were consistently elevated in Cd-treated chicks, with the exception of those also maintained on diets low in Ca. The results indicate that cadmium toxicity exerts at least two effects on Ca metabolism, one at the intestinal level and another at the level of the bone, kidney, or both.