Identification of the calcium-sensing receptor in the developing tooth organ.

Calcium (Ca2+) is a critical component of tooth enamel, dentin, and the surrounding extracellular matrix. Ca2+ also may regulate tooth formation, although the mechanisms for such action are poorly understood. The Ca2+-sensing receptor (CaR) that is expressed in the parathyroid gland, kidney, bone, and cartilage has provided a mechanism by which extracellular Ca2+ can regulate cell function. Because these tissues play an important role in maintaining mineral homeostasis and because Ca2+ is hypothesized to play a crucial role in tooth formation, we determined whether the CaR was present in teeth. In this study, using immunohistochemistry, CaR protein was detected in developing porcine molars localized in the predentin (pD), early secretory-stage ameloblasts, maturation-stage smooth-ended ameloblasts (SA), and certain cells in the stratum intermedium. CaR protein and messenger RNA (mRNA) were detected also in an immortalized ameloblast-like cell line (PABSo-E) using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and Northern analysis. Based on the observation that the CaR is expressed in cultured ameloblasts, we determined whether increments in medium Ca2+ concentration could activate the intracellular Ca2+ signal transduction pathway. In PABSo-E cells, increasing extracellular Ca2+ in the medium from 0 (baseline) to 2.5mM or 5.0 mM resulted in an increase in intracellular Ca2+ above baseline to 534 +/- 69 nM and 838 +/- 86 nM, respectively. Taken together, these results suggest that the CaR is expressed in developing teeth and may provide a mechanism by which these cells can respond to alterations in extracellular Ca2+ to regulate cell function and, ultimately, tooth formation.

Divalent cations modulate PTH-dependent 3',5'-cyclic adenosine monophosphate production in renal proximal tubular cells.

The kidney and parathyroid gland play key roles in calcium (Ca++) homeostasis. Recent data suggest that the kidney, in addition to being a primary target for PTH, also recognizes changes in the concentration of extracellular Ca++, thereby modulating hormone-dependent cAMP production, 1,25-dihydroxyvitamin D synthesis, and renin secretion. In this study, we examined: 1) the effects of varying concentration of divalent cations on PTH-dependent cAMP production in renal proximal tubular cells; and 2) the mechanisms by which extracellular Ca++ exerts its inhibitory effects on cAMP production. Single cell suspensions composed of 80-90% proximal tubular cells were prepared from cortical homogenates by collagenase digestion and sieving. In the presence of 1 mM isobutylmethylxanthine, cAMP content was measured by RIA in 5-15 min incubations and showed a 5- to 6-fold increase in response to PTH (10(-11) -10(-6) M). Increasing extracellular Ca++ and magnesium (Mg++) from 0 and 0.5 mM, respectively, to 5.0 mM inhibited PTH-dependent (3 x 10(-9) M) cAMP production by 54 +/- 4% and 47 +/- 6%, respectively. The half maximal inhibitory concentration for both Ca++ and Mg++ was 0.9 mM. In addition, increasing extracellular barium (Ba++) or strontium (Sr++) from 0-10 mM inhibited PTH-dependent (3 x 10(-9) M) production by 54 +/- 7% and 62 +/- 6% with half of the maximal observed inhibition at 2.2 and 2.7 mM, respectively. The inhibition of PTH-dependent cAMP production by 2.5 mM Ca++ was not reversed by the calcium channel blockers diltiazem or verapamil (10(-4) M). However, changes in intracellular calcium may play some role in the inhibitory effects of Ca++ on cAMP production, since ionomycin (10(-6)-10(-5) M) lowered PTH-dependent cAMP production by 25-36%. Our data suggest that the proximal tubular cell can sense physiologically relevant changes in Ca++, providing a potential mechanism for the modulation of 1,25-dihydroxyvitamin D production or other tubular functions relevant to fluid and mineral homeostasis.

Hyperammonemia in urea cycle disorders: role of the nephrologist.

Hyperammonemia associated with inherited disorders of amino acid and organic acid metabolism is usually manifested by irritability, somnolence, vomiting, seizures, and coma. Although the majority of these patients present in the newborn period, they may also present in childhood, adolescence, and adulthood with failure to thrive, persistent vomiting, developmental delay, or behavioral changes. Persistent hyperammonemia, if not treated rapidly, may cause irreversible neuronal damage. After the diagnosis of hyperammonemia is established in an acutely ill patient, certain diagnostic tests should be performed to differentiate between urea cycle defects and other causes of hyperammonemic encephalopathy. In a patient with a presumed inherited metabolic disorder, the aim of therapy should be to normalize blood ammonia levels. Recent experience has provided treatment guidelines that include minimizing endogenous ammonia production and protein catabolism, restricting nitrogen intake, administering substrates of the urea cycle, administering compounds that facilitate the removal of ammonia through alternative pathways, and, in severe cases, dialysis therapy. Initiation of dialysis in the encephalopathic patient with hyperammonemia is indicated if the ammonia blood level is greater than three to four times the upper limit of normal. Hemodialysis is the most effective treatment for rapidly reducing blood ammonia levels. Continuous hemofiltration and peritoneal dialysis are also effective modalities for reducing blood ammonia levels. An improved understanding of the metabolism of ammonia and neurological consequences of hyperammonemia will assist the nephrologist in providing optimal care for this high-risk patient population.

X-linked laterality sequence: situs inversus, complex cardiac defects, splenic defects.

The association of abdominal situs inversus, complex cardiac defects, and alterations in development of the spleen represents a developmental field complex with variable expression of altered laterality. Familial and inherited cases documenting respectively autosomal recessive and dominant inheritance have been reported. We report on the first family in which X-linked recessive inheritance of this defect has been documented.

Chronic anemia as a complication of parvovirus B19 infection in a pediatric kidney transplant patient.

This is a report of unexplained anemia that persisted for 4 months in an adolescent renal transplant patient receiving immunosuppression that included prednisone, tacrolimus, and mycophenolate mofetil. This patient required monthly blood transfusions for fatigue, palpitations, and hematocrit levels between 15% and 17%. In addition, his posttransplant course was notable for the development of insulin-dependent diabetes mellitus. While receiving low-dose prednisone, he was switched from tacrolimus to cyclosporin and tapered off insulin injections over the next 2 months. At 4.5 months post-transplantation, further diagnostic evaluation was suggestive of parvovirus B19 infection as the cause for our patient's chronic anemia. After testing negative for serum-specific parvovirus B19 IgM and IgG antibodies, parvovirus B19 infection was detected in blood by the polymerase chain reaction. Treatment with intravenous immunoglobulin (1 g/kg per day x 2 days) resulted in normalization of both his reticulocyte count and hematocrit within 6 weeks. At 4 months after receiving the immunoglobulin infusion, he has maintained a normal hematocrit level and stable renal function without requiring further blood transfusions.

Rickets in an infant with Williams syndrome.

Calcium homeostasis is altered in patients with Williams syndrome. We report an infant in whom Williams syndrome was diagnosed at 4 weeks who presented with hypercalcemia, hypercalciuria, and medullary nephrocalcinosis. Fluorescence in situ hybridization demonstrated a deletion of the elastin gene on chromosome 7. This infant was treated with a low-calcium/vitamin D-deficient infant formula that resulted in the development of rickets. Replacement of the low-calcium/vitamin D-deficient formula with standard formula led to resolution of the rickets.

Impaired natriuresis after volume expansion in the aged rat.

These experiments were designed to compare the natriuretic ability of old(age 22-24 months) and young (4-6 months) rats after volume expansion. No difference in extracellular fluid volume was noted as estimated by inulin space; old 18.8 +/- 0.6% and young 18.2 +/- 0.7% of body weight. Standard clearance techniques were utilized in unanesthetized animals. The fraction of infused sodium excreted during and after expansion with isotonic saline equal to 7% BW was statistically lower in the old group 53 +/- 2 vs. 68 +/- 3% (p less than 0.01). Similar measurements were made during the infusion of whole blood equal to 2.3% BW. Again the old rats excreted a significantly lower fraction of the infused Na, 55 +/- 10 vs. young 112 +/- 12%. These differences do not appear to be explained by changes in glomerular filtration rate, blood pressure, hematocrit or serum protein concentration. We conclude that aged rats have an impaired ability to excrete sodium with volume expansion but the mechanism for this defect is yet to be determined.

Increased fluoride content in the femur growth plate and cortical bone of uremic rats.

In chronic renal insufficiency (CRI), serum levels of fluoride (F-) are elevated. However, there is limited information about the effects of F- on bone in CRI. In this study, we determined whether F- content in mineralizing tissue (growth plate, cortical bone, and bone marrow of the femur) is affected by uremia. Adult rats were divided into two groups [sham-operated (S) and 5/6 nephrectomized (Nx)]. At sacrifice, the serum creatinine (mg/dl) in the S and 5/6 Nx animals was 0.37+/-0.09 (mean+/-SD) and 1.10+/-0.34 at 4 weeks, and 0.38+/-0.04 and 0.90+/-0.36 at 8 weeks, respectively. The serum calcium, phosphorus, and parathyroid hormone levels were lower and the serum 1, 25-dihydroxyvitamin D levels were higher in S animals than Nx animals at both 4 and 8 weeks. F- urinary excretion (ppm/24 h) was reduced in Nx animals at 4 weeks (34.0+/-19.2) versus S animals (50.7+/-12.9) (P<0.05). F content (ppm) was significantly increased in the growth plate in Nx animals compared with S animals both at 4 weeks (550+/-167 vs. 353+/-63) and at 8 weeks (654+/-135 vs. 396+/-97), respectively (P<0.01). The F- content in cortical bone was similarly increased in Nx animals compared with S animals, but was only statistically increased at 8 weeks. There was no difference in bone marrow F- content between the two groups. In conclusion, this study suggests that in CRI, there is a rapid increase in F- content of the distal femur in the growth plate region, with a subsequent slower increase of F- content in cortical bone.

Successful treatment with hydrochlorothiazide and amiloride in an infant with congenital nephrogenic diabetes insipidus.

We report a 9-month-old male Latino infant with congenital nephrogenic diabetes insipidus (NDI) who presented with hypernatremic dehydration aggravated by severe gastroenteritis. Initially, the infant was managed with intravenous fluids followed by standard 20 cal/ounce formula and pharmacological therapy, resulting in normalization of his serum sodium level. While hydrochlorothiazide therapy alone or in combination with prostaglandin inhibitors or amiloride has been successful in children and adolescents, this is the first report of the successful use of hydrochlorothiazide and amiloride in an infant with congenital NDI.

Endogenous thromboxane A2 does not contribute to the contractile response of human umbilical artery strips to 5-hydroxytryptamine.

1. To investigate the possible role of endogenous thromboxane A2 (TXA2) in 5-hydroxytryptamine (5-HT)-induced contraction, human umbilical artery strips were suspended in isolated organ chambers for measurement of isometric force. 2. In endothelium intact strips, arachidonic acid (AA;1 microM) potentiates the contractile response to 5-HT, whereas the response was reduced by indomethacin (INDO;10 microM). De-endothelialized strips showed reduced responses to 5-HT. 3. Arachidonic acid-induced potentiation of the responses to 5-HT was prevented by INDO, and the TXA2 synthase inhibitor dazoxiben (DAZ;1 microM and 10 microM) was without effect on the responses to 5-HT in endothelium intact strips. 4. Taken collectively, these results suggest that, in human umbilical artery strips, the contractile response to 5-HT is at least partly dependent on the 5-HT induced release of an endothelium-derived contracting factor (EDCF), which is a cyclooxygenase metabolite. The lack of effect of DAZ indicates that TXA2 is not the EDCF released during the contractile response of human umbilical artery strips to 5-HT.

Non-capacitative calcium entry in Chinese hamster ovary cells expressing the platelet-derived growth factor receptor.

Platelet-derived growth factor (PDGF) is believed to produce intracellular calcium (Ca2+i) transients by inositol trisphosphate (InsP3)-mediated release of intracellular Ca2+ stores followed by "capacitative" Ca2+ entry due to emptying of these stores. We examined the roles for the phospholipase Cgamma-InsP3 pathway and the emptying of InsP3-dependent intracellular Ca2+ stores in PDGF-mediated Ca2+ entry. Intracellular Ca2+ release and Ca2+ entry were measured with fluorometric methods in Chinese hamster ovary cells expressing wild type or mutant PDGF receptors. Activation of the wild type PDGF receptor caused both intracellular "Ca2+ release, " measured in nominally 0 Ca2+ extracellular medium, and "Ca2+ entry, " measured upon addition of 2 mM Ca2+ medium. Both phases were absent in Chinese hamster ovary cells expressing a PDGF receptor mutant (Y977F,Y989F) that fails to bind phospholipase Cgamma. Blockade of the InsP3 receptor, by microinjection of single cells with low molecular weight heparin (5-50 mg/ml), blocked only Ca2+i release (following PDGF or flash photolysis of caged InsP3) and had no effect on PDGF-induced Ca2+ entry. In whole cell patch-clamp experiments, intracellular heparin also failed to block PDGF-evoked ion currents. Release of InsP3-dependent intracellular Ca2+ stores, by flash photolysis of caged InsP3, was apparently not sufficient to maximally activate Ca2+ entry. Intracellular InsP3 caused significantly less Ca2+ entry than PDGF alone. These data suggest that InsP3 alone is not sufficient to maximally activate Ca2+ entry by the capacitative pathway and that products of phosphatidylinositol 4,5-bisphosphate breakdown other than InsP3 probably play a role in PDGF-mediated Ca2+ entry.

Effects of simulated myocardial ischemic conditions on the responses of isolated human umbilical artery strips to 5-hydroxytryptamine and prostaglandin F2 alpha.

1. The effects of simulated myocardial ischemic conditions on the contractile response of isolated human umbilical artery (HUA) strips to 5-hydroxytryptamine (5-HT) and prostaglandin F2 alpha (PGF2 alpha) were studied. 2. During simulated myocardial ischemic conditions the contractile response of HUA strips to 5-HT was lower than the response to the monoamine under oxygenated conditions. Under simulated ischemic conditions the response to 5-HT was further depressed by the cyclooxygenase inhibitor indomethacin (10 microM) and increased by the NO synthase inhibitor L-NAME (100 microM). 3. During simulated ischemic conditions the response of the HUA to a submaximal concentration of PGF2 alpha (3 microM) was reduced. Indomethacin (10 microM) further reduced the response to the prostanoid whereas L-NAME (100 microM) enhanced the response to PGF2 alpha. 4. It is concluded that during simulated myocardial ischemic conditions lactate negatively modulates the contractile response of HUA strips to 5-HT. Apparently, during simulated myocardial ischemic conditions in the HUA the production and/or release of EDRF/NO was not affected.

Urinary concentrating defect in the aged rat.

The renal concentrating ability of Fischer 344 rats was studied at 23 and 4 mo of age. Maximum urine concentration after 40 h of dehydration with or without vasopressin injection was significantly lower (P less than 0.01) in old (2,550 +/- 70 and 2,363 +/- 107 mosmol/kg H2O2, respectively) vs. young (3,242 +/- 50 and 3,162 +/- 50 mosmol/kg H2O, respectively) rats. Free water reabsorption (TcH2O/GFR) rose progressively as a function of osmolar clearance, and at similar values of distal solute delivery TcH2O was clearly reduced in the old group. Free water formation (CH2O/GFR) rose linearly as a function of urine flow and was not different between old and young rats. Glomerular filtration rate was also not different between age groups under the conditions studied. Nonurea (sodium + potassium + ammonium) x 2 and urea solute concentrations as well as total calculated osmolality in the cortex, outer medulla, or inner medulla were not different between age groups. Because the indices of ascending limb solute delivery and transport and the solute gradient for water reabsorption were similar, we conclude that the concentrating defect in aged rats is most likely secondary to a decrease in water permeability along the collecting duct.

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha-thrombin.

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha-thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha-thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha-thrombin receptors may have a second, alternative mechanism for activating CA2+i release.

Reduced expression of the renal calcium-sensing receptor in rats with experimental chronic renal insufficiency.

Chronic renal insufficiency is associated with elevated serum parathyroid hormone (PTH) levels (2 degrees HPT), deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D), and hypocalciuria. In chronic renal insufficiency, the 2 degrees HPT may result from reduced expression of the parathyroid gland extracellular Ca(2+)-sensing receptor (CaSR). Since the CaSR was cloned from rat and human kidney, this study examined in rats whether expression of the renal CaSR is altered in experimental chronic renal insufficiency. Four weeks after chronic renal insufficiency was induced by 5/6 nephrectomy (Nx) in Sprague Dawley rats, the serum creatinine concentration was 0.96+/-0.06 mg/dl compared with 0.35+/-0.02 mg/dl in sham-operated animals (P < 0.05). The serum total Ca2+ and phosphorus concentrations were not different. In the Nx group, the serum concentration of amino-PTH was higher (65+/-8 pg/ml), and the concentration of 1,25(OH)2D was significantly lower (47+/-5 pg/ml) compared with 45+/-5 pg/ml and 61+/-4 pg/ml (P = 0.05) in the sham group, respectively. In a subset of rats studied, the Nx group was hypocalciuric (1.4+/-0.5 mg/kg per d) compared with the sham group (3.7+/-0.5 mg/kg per d) (P < 0.05). In the Nx rats, CaSR mRNA expression and CaSR protein levels were found to be reduced by 35 and 38%, respectively, than those observed in controls. These results suggest that reduced renal CaSR expression in chronic renal insufficiency may play a role in disordered mineral ion homeostasis, including hypocalciuria.

Hypoxia and response of human umbilical artery strips to 5-hydroxytryptamine: role of prostaglandin F2 alpha.

1. This article examines the effects of hypoxia on the contractile response of isolated human umbilical artery strips to 5-hydroxytryptamine (5-HT). 2. Hypoxic conditions produce a large increase in the contractile response to 5-HT without a significant alteration of the sensitivity evaluated at the level of the pD2 value. Indomethacin (10 microM) reduced hypoxia-induced potentiation of the response to 5-HT and decreased the response to the monoamine under oxygenated conditions. 1-NAME (100 microM) did not further increase the effect of hypoxia on the vessel response to 5-HT and increased the response to 5-HT under oxygenated conditions. 3. Taken together, these results suggest that, at least partially, the response of human umbilical artery strips to 5-HT depends on 5-HT release of a contracting prostanoid which is a product of the cyclooxygenase pathway. Furthermore, during hypoxia in human umbilical artery strips, there appears to be impairment of the basal production and/or release of EDRF/NO. 4. A subthreshold concentration of prostaglandin F2 alpha (1 nM) potentiates the response to 5-HT in indomethacin-pretreated umbilical artery strips. The data raise the possibility that prostaglandin F2 alpha might be the prostanoid released during hypoxia, which in turn potentiates the response of the human umbilical artery to 5-HT.

Cesarea na situacao transversa. / Cesarean section in the case of transverse lie

Os autores analisam o prognostico materno-fetal nos casos de situacao transversa submetidos a cesarea. Foram estudados 109 casos de situacao transversa, dos quais cinco eram nuliparas (4,6%), 54 multiparas (49,5%) e 50 grandes multiparas (45,9%). A idade gestacional variou de 29 a 42 semanas; a bolsa encontrava-se integra em 63 casos (57,8%) e rota em 46 (42,3%). Os fetos estavam vivos em 104 casos (95,4%) e mortos em cinco (2,6%) no momento da internacao. A incisao uterina foi em 107 vezes a segmentar transversa (98,1%) e em sete oportunidades em "T" invertido, devido a dificuldade de extracao fetal. A raquianestesia foi utilizada 91 vezes (83,5%), a anestesia geral em 12 casos (11%) e a raquidea mais geral em seis casos (5,5%). O trauma fetal mais frequente foi a equimose ocorrendo seis vezes (66,7%); a paresia do membro superior, paralisia branquial e a fratura do umero ocorreram uma unica vez (11,1%).Ocorreram sete obitos no periodo neonatal, todos recem-natos com peso inferior a 2.500 g. Foram identificados 11 casos de morbidade materna