Pharmacokinetics of magnesium and its effects on clinical variables following experimentally induced hypermagnesemia

The objectives of this study were to describe pharmacokinetic and pharmacodynamic changes as a result of a single intravenous administration of magnesium sulfate (MgSO₄) to healthy horses. MgSO₄ is a magnesium salt that has been used to calm horses in equestrian competition and is difficult to regulate because magnesium is an essential constituent of all mammals. Six healthy adult female horses were administered a single intravenous dose of MgSO₄ at 60 mg/kg of body weight over 5 min. Blood, urine, and cerebrospinal fluid (CSF) samples were collected, and cardiovascular parameters were monitored and echocardiograms performed at predetermined times. Noncompartmental pharmacokinetic analysis was applied to plasma concentrations of ionized magnesium (Mg²⁺). Objective data were analyzed using the Wilcoxon rank-sum test with p < .05 used as a determination for significance. Plasma concentrations of Mg²⁺ increased nearly fivefold, ionized calcium (Ca²⁺) decreased by nearly 10%, and the Ca²⁺ to Mg²⁺ ratio declined more than 3.5-fold and remained different than baseline until 24 hr (p < .05). Significant changes were seen with urinary fractional excretion of electrolytes, cardiovascular parameters, and echocardiographic measurements. No changes were detected in CSF electrolyte concentrations. The decrease in Ca²⁺ result of hypermagnesemia supports the interaction between these cations. Alterations detected in plasma electrolyte concentrations and urinary fractional excretion of electrolytes may serve as biomarkers for regulatory control for the nefarious administration of MgSO₄.     

Evaluation of an Electrolyte Analyser for Measurement of Concentrations of Ionized Calcium and Magnesium in Cats

The goal of this study was to evaluate the Nova CRT 8 electrolyte analyser for determination of concentrations of ionized calcium (Ca_i) and magnesium (Mg_i) in cats, todetermine the effects of sample handling and storage and to establish reference ranges. The precision and analytical accuracy of the Nova CRT 8 analyser were good. The concentrations of Ca_i and Mg_i were significantly lower in aerobically handled serum samples than in those handled anaerobically. The concentrations of Ca_i and Mg_i differed significantly among whole blood, plasma and serum. In anaerobically handled serum, the concentration of Ca_i was stable for 8 h at 22°C, for 5 days at 4°C and for 1 week at −20°C. The concentration of Mg_i was stable for 4 h at 22°C but for less than 24 h at 4°C and for less than 1 week at −20°C. In serum from 36 cats, the reference ranges were 1.20–1.35 mmol/L for Ca_i and 0.47–0.59 mmol/L for Mg_i. The Nova CRT 8 electrolyte analyser is suitable for determination of Ca_i and Mg_i concentrations in cats. Anaerobically handled serum samples are recommended and, stored at room temperature, they yield accurate results when analysed within 4 h.     

Parathyroid hormone-related protein and calcium concentrations in milk and blood of ewes

Parathyroid hormone-related protein (PTHrP) and calcium (Ca) concentrations were measured 2 to 3 months postpartum in milk and plasma or serum of 22 ewes by the use of commercial radioimmunoassay kits for PTHrP concentration, colorimetry for serum total Ca concentration (Ca_tot), and atomic absorption spectrophotometry for milk total Ca concentration. Scatter plots did not reveal dependency between milk Ca and Ca_tot, Ca_tot and plasma PTHrP, milk Ca and milk PTHrP, and milk PTHrP and plasma PTHrP. Thus, the systemic and mammary effects of PTHrP are not major determinants of the endocrine, paracrine, and autocrine regulation of Ca homeostasis during lactation in ewes.     

The effects of surgery and anesthesia on blood magnesium and calcium concentrations in canine and feline patients

OBJECTIVE: To demonstrate the effect of anesthesia and surgery on serum ionized magnesium and ionized calcium concentrations in clinical canine and feline patients. ANIMALS: 37 client-owned dogs, ASA PS I-III and 10 client-owned cats, ASA PS I, all receiving anesthesia for elective or emergent surgery at a Veterinary Teaching Hospital. MATERIALS AND METHODS: Plasma ionized and serum total magnesium, and plasma ionized calcium were measured prior to and after a group-standardized anesthetic protocol. RESULTS: Regardless of pre-operative medication (hydromorphone or butorphanol), anesthetic induction (thiopental or lidocaine/hydromorphone/diazepam (LHD) and propofol combination), or type of surgical procedure (peripheral surgery or laparotomy), post-operative plasma ionized calcium concentration decreased in all groups of dogs, while post-operative plasma ionized magnesium increased in all groups, although the changes were not always significant. The dogs who were induced with an LHD and propofol technique had a greater increase in ionized magnesium (0.36 +/- 0.07 to 0.42 +/- 0.07 mmol L(-1)) than the group in which anesthesia was induced with thiopental (0.41 +/- 0.07 to 0.42 +/- 0.07 mmol L(-1), p = 0.009). The cats showed similar changes in ionized magnesium and ionized calcium, and also had a significant increase in serum total magnesium (2.17 +/- 0.20 to 2.31 +/- 0.25 mg dL(-1), p = 0.009) CONCLUSIONS, CLINICAL RELEVANCE: A post-operative decrease in ionized calcium was demonstrated in healthy animals, as well as an increase in ionized or total magnesium after various anesthetic protocols and surgeries. These changes, while statistically significant, do not appear to be clinically significant, as values remained within reference ranges at all times.     

pH-dependent modulation of intracellular free magnesium ions with ion-selective electrodes in papillary muscle of guinea pig

A change in pH can alter the intracellular concentration of electrolytes such as intracellular Ca²⁺ and Na⁺ ([Na⁺]_i) that are important for the cardiac function. For the determination of the role of pH in the cardiac magnesium homeostasis, the intracellular Mg²⁺ concentration ([Mg²⁺]_i), membrane potential and contraction in the papillary muscle of guinea pigs using ion-selective electrodes changing extracellular pH ([pH]_o) or intracellular pH ([pH]_i) were measured in this study. A high CO₂-induced low [pH]_o causes a significant increase in the [Mg²⁺]_i and [Na⁺]_i, which was accompanied by a decrease in the membrane potential and twitch force. The high [pH]_o had the opposite effect. These effects were reversible in both the beating and quiescent muscles. The low [pH]_o-induced increase in [Mg²⁺]_i occurred in the absence of [Mg²⁺]_o. The [Mg²⁺]_i was increased by the low [pH]_i induced by propionate. The [Mg²⁺]_i was increased by the low [pH]_i induced by NH₄Cl-prepulse and decreased by the recovery of [pH]_i induced by the removal of NH₄Cl. These results suggest that the pH can modulate [Mg²⁺]_i with a reverse relationship in heart, probably by affecting the intracellular Mg²⁺ homeostasis, but not by Mg²⁺ transport across the sarcolemma.     

Mechanisms involved in the cytosolic calcium ion elevation induced by activating factors in chicken and rat phagocytes

The aim of the present study was to determine the mechanism of cytosolic calcium ion concentration [Ca²⁺]_i elevation in chicken and rat phagocytes stimulated with phorbol myristate acetate (PMA), leukotriene B₄ (LTB₄), formyl‐methionyl‐leucyl‐phenylananine (fMLP) and Saccaromyces cerevisiae culture supernatant (SCS). Pretreatment with EGTA completely suppressed the PMA‐induced [Ca²⁺]_i elevation in rat and chicken phagocytes, suggesting that all the [Ca²⁺]_i elevation induced in the PMA‐stimulated rat and chicken phagocytes was attributable to the influx of extracellular Ca²⁺. On the other hand, the elevation of LTB₄‐, FMLP‐ and SCS‐induced [Ca²⁺]_i was only partially suppressed by ethyleneglycol‐bis (β‐aminoethyl)‐N,N,N′,N′‐tetraacetic acid ethylene (EGTA) pretreatment of phagocytes. The results indicated that two pathways of [Ca²⁺]_i elevation, recruitment from the intracellular Ca²⁺ store and influx of extracellular Ca²⁺, are involved in the [Ca²⁺]_i elevation of LTB₄‐, fMLP‐ and SCS‐stimulated phagocytes. In fMLP‐stimulated rat neutrophils, [Ca²⁺]_i elevation showed a two‐phase pattern in which the time lag between the first and second phase was approximately 1 min. The EGTA treatment of the fMLP‐stimulated cells induced a reduction of the first phase level and a disappearance of the second phase. The reason for the special influence of EGTA observed in fMLP‐stimulated cells is unknown, but the disappearance of the second phase of the [Ca²⁺]_i may be elicited by the EGTA‐induced decrease of the first phase [Ca²⁺]_i elevation that depends on IP₃ and diacylglycerol induced by fMLP.     

Effects of 1,25-dihydroxyvitamin D3 on calcium and phosphorus homeostasis in sheep fed diets either adequate or restricted in calcium content

It was the aim of the present study to collect basic data on calcium (Ca) and phosphorus (P) homoeostasis in sheep. Two series of experiments were carried out to investigate the effects of 1,25-dihydroxyvitammin D₃ (calcitriol) in supraphysiological dosage in combination with varying alimentary Ca supply. In the first series, blood samples were collected over 72 h to determine the concentrations of total Ca (Ca), ionized Ca (Ca²⁺), inorganic phosphate (P_i), and the bone resorption marker CrossLaps (CL). In the second series, measurements were carried out over 12 h. In addition, urine samples were collected to calculate the fractional excretions (FE) of Ca and P_i. Changes in plasma macromineral concentrations (P < 0.01) as well as in CL (P < 0.001) and endogenous calcitriol (P < 0.05) were observed in the alimentary Ca-restricted animals, indicating that the reduction of daily Ca intake challenged the animals’ macromineral homeostatic mechanisms. However, the Ca-restricted diet had an effect on neither FE of Ca nor on FE of P_i. The treatment resulted in peak serum calcitriol concentrations between 1,900 and 2,500 pg/mL, and supraphysiological concentrations were maintained for the next 48 h. Irrespective of dietary Ca, calcitriol had hypercalcemic and hyperphosphatemic effects. An increase in CL was revealed only in the Ca-restricted, calcitriol-treated sheep (P < 0.01), reflecting a remarkable enhancement of Ca mobilization from the bone by calcitriol exclusively in this group. From these data, it can be concluded that the sheep can be a suitable animal model for studying catabolic effects of Ca deficiency and calcitriol on bone metabolism.     

Hypocalcemia in a critically ill patient

Objective: To present a case of clinical hypocalcemia in a critically ill septic dog. Case summary: A 12‐year old, female spayed English sheepdog presented in septic shock 5 days following hemilaminectomy surgery. Streptococcus canis was cultured from the incision site. Seven days after surgery, muscle tremors were noted and a subsequent low serum ionized calcium level was measured and treated. Intensive monitoring, fluid therapy, and antibiotic treatment were continued because of the sepsis and hypocalcemia, but the dog was euthanized 2 weeks after surgery. New or unique information provided: Low serum ionized calcium levels are a common finding in critically ill human patients, especially in cases of sepsis, pancreatitis, and rhabdomyolysis. In veterinary patients, sepsis or streptococcal infections are not commonly thought of as a contributing factor for hypocalcemia. Potential mechanisms of low serum ionized calcium levels in critically ill patients include intracellular accumulation of calcium ions, altered sensitivity and function of the parathyroid gland, alterations in Vitamin D levels or activity, renal loss of calcium, and severe hypomagnesemia. Pro‐inflammatory cytokines and calcitonin have also been proposed to contribute to low ionized calcium in the critically ill. Many veterinarians rely on total calcium levels instead of serum ionized calcium levels to assess critical patients and may be missing the development of hypocalcemia. Serum ionized calcium levels are recommended over total calcium levels to evaluate critically ill veterinary patients.     

Bovine urolithiasis in Andhra Pradesh

Urinary calculi from three bulls, six bullocks and one male buffalo from the Guntur district were investigated. By chemical analysis only CO₃², Ca²⁺, Mg²⁺, and a trace of PO¾^- were found. X-ray analysis showed that all samples consisted of magnesian calcite with an atomic percentage of magnesium between 14.5 and 21.9. The size of the crystallites varied approx. from 200 to 400 Å.     

Salt-Lick–Induced Soil Disturbance in the Teton Wilderness,USA

Manmade salt licks on public lands throughout the Rocky Mountain West have been created to attract large game for hunting purposes. This practice is both illegal and controversial, but is of particular importance in otherwise pristine wilderness landscapes. The impact of widespread saltlicks on public lands has never been quantified. This study was undertaken to examine the degree of change in soil physical and chemical properties caused by approximately 10–60 years of salt application in the Teton Wilderness of Wyoming, USA. A total of 27 sites were identified, surveyed, and paired with non–salt-affected control areas. Three replicate sampling points were located within each salt site and in each of the paired control areas. Soil samples from each site were analyzed for soil bulk density, soil salinity as electrical conductance (EC), pH, organic matter content, sodium absorption ratio (SAR), and exchangeable concentrations of sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), and magnesium (Mg²⁺). Salt-treated site centers were found to have elevated EC, bulk density, pH, SAR, and Na⁺ concentration compared to the no-salt controls. Salt-affected sites also contained decreased organic matter contents and decreased concentrations of Ca²⁺ and Mg²⁺. Observed differences were due to the addition of Na⁺ to the soil solum as well as direct effects of ungulates. Soil compaction appears to have a greater impact on plant establishment than the actual presence of NaCl. Salt licks established in wilderness areas habituate animals to localized zones causing extensive soil trampling and consumption of surface soils by grazing ungulates.     

Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis

OBJECTIVE: To evaluate calcium balance and parathyroid gland function in healthy horses and horses with enterocolitis and compare results of an immunochemiluminometric assay (ICMA) with those of an immunoradiometric assay (IRMA) for determination of serum intact parathyroid hormone (PTH) concentrations in horses. ANIMALS: 64 horses with enterocolitis and 62 healthy horses. PROCEDURES: Blood and urine samples were collected for determination of serum total calcium, ionized calcium (Ca2+) and magnesium (Mg2+), phosphorus, BUN, total protein, creatinine, albumin, and PTH concentrations, venous blood gases, and fractional urinary clearance of calcium (FCa) and phosphorus (FP). Serum concentrations of PTH were measured in 40 horses by use of both the IRMA and ICMA. RESULTS: Most (48/64; 75%) horses with enterocolitis had decreased serum total calcium, Ca2+, and Mg2+ concentrations and increased phosphorus concentrations, compared with healthy horses. Serum PTH concentration was increased in most (36/51; 70.6%) horses with hypocalcemia. In addition, FCa was significantly decreased and FP significantly increased in horses with enterocolitis, compared with healthy horses. Results of ICMA were in agreement with results of IRMA. CONCLUSIONS AND CLINICAL RELEVANCE: Enterocolitis in horses is often associated with hypocalcemia; 79.7% of affected horses had ionized hypocalcemia. Because FCa was low, it is unlikely that renal calcium loss was the cause of hypocalcemia. Serum PTH concentrations varied in horses with enterocolitis and concomitant hypocalcemia. However, we believe low PTH concentration in some hypocalcemic horses may be the result of impaired parathyroid gland function.     

Parturient Paresis in the Cow: Serum Ionized Calcium Concentrations Before and after Treatment with Different Calcium Solutions – Classification of Different Degrees of Hypo- and Hypercalcemia

Serum ionized calcium concentrations (Ca_F) were determined in 87 Swedish red-and-white cows and 10 Swedish Friesian cows with clinical signs of parturient paresis. All cows were in the week prior to or after parturition. A classification of the severity of hypocalcemia in terms of serum ionized calcium was devised. Eight cows had normal serum ionized calcium concentrations (Cap 1.06–1.26 mmol/1); 15 had slight (Ca_F 0.80–1.05 mmol/1); 43 a moderate (Ca_F 0.50–0.79 mmol/1), and 31 asevere (Ca_F < 0.50 mmol/1) hypocalcemia.All cows were given 8 or 8.3 g of calcium intravenously. Of 8 normocalcemic cows 7 (87.5 %) reached a maximum posttreatment serum ionized calcium concentration > 1.80 mmol/1 (severe hypercalcemia). This was also found in 13 of 15 (86.7 %) slightly hypocalcemic cows and in 31 of 43 (72.1 %) moderately hypocalcemic cows. In the severe hypocalcemia group 14 of 31 (45.2 %) had maximum posttreatment Cap > 1.80 mmol/1).These findings emphazise the need of a rapid pretreatment evaluation of the degree of hypocalcemia. The present study also underlined the difficulty in predicting serum ionized calcium from serum total calcium concentrations.     

Serum concentrations of parathyroid hormone and 25-OH vitamin D3 in cats with urethral obstruction

Objective: To measure serum parathyroid hormone (PTH) and 25‐hydroxyvitamin D₃ concentrations in cats with urethral obstruction. Design: Prospective single cohort study. Setting: University affiliated veterinary teaching hospital. Animals: Male cats with urethral obstruction. Interventions: Routine blood samples drawn from male cats with urethral obstruction. Measurements and main results: Measured variables included blood gas parameters, plasma sodium, potassium, chloride, and ionized calcium concentrations, as well as serum blood urea nitrogen, creatinine, phosphorus, PTH, and 25‐hydroxyvitamin D₃ concentrations. PTH was inversely correlated with ionized calcium and positively correlated with serum phosphorus. No discernable relationship could be found between 25‐hydroxyvitamin D₃ and any of the measured parameters. Conclusions: Lack of parathyroid response does not appear to be the underlying mechanism for ionized hypocalcemia in cats with urethral obstruction.     

Effects of aerobic and anaerobic fluid collection on biochemical analysis of peritoneal fluid in healthy horses and horses with colic

Objective: To determine whether in healthy horses and those with colic, exposure of peritoneal fluid to room air affects values obtained on biochemical analysis. Study Design: Prospective study. Animals: Adult horses with a primary complaint of acute abdominal pain (n=29) and 12 healthy horses. Methods: Peritoneal fluid was aseptically collected under aerobic and anaerobic conditions. After collection, pH, PCO₂, PO₂, HCO₃^?, Na⁺, ionized Ca²⁺, K⁺, lactate, and glucose were immediately measured using a commercial blood gas analyzer. Biochemical variables were compared between aerobically and anaerobically obtained samples using a paired t‐test. Results: In healthy horses, peritoneal fluid samples collected under anaerobic conditions had higher PCO₂ and ionized Ca²⁺ and lower PO₂, HCO₃^?, and pH compared with samples exposed to air. No differences were observed for K⁺, Na⁺, glucose, and lactate. In horses with colic, samples collected anaerobically had higher PCO₂, ionized Ca²⁺, Na⁺, and glucose and lower PO₂, HCO₃^?, and pH value compared with samples exposed to air. No differences were observed for K⁺ and lactate. Conclusion: Exposure of peritoneal fluid to room air had a significant effect on pH, PCO₂, PO₂, and variables associated or dependent on changes in pH such as HCO₃^? and ionized Ca²⁺. Interpretation of biochemical analysis of peritoneal fluid may be influenced by sample collection method.     

Validation of the Nova CRT8 for the measurement of ionized magnesium in feline serum

Background: Evaluation of serum magnesium (Mg) concentration is becoming important in human and veterinary critical care medicine. An ion‐selective electrode can measure the physiologically active ionized fraction. Objectives: The purpose of this study was to validate an ion‐specific electrode analyzer and assay for measuring ionized Mg in feline serum and to determine a reference interval for this analyte in cats. Methods: Venous blood samples were collected anaerobically from clinically healthy cats, and the serum was used to validate the analyzer and assay. This included investigating the stability of samples stored at different temperatures, intra‐ and interassay precision, linearity, analytical sensitivity, and potential interferences from bilirubin, lipemia, hemoglobin, or serum separator tubes. A reference interval was calculated. Results: Serum samples evaluated for ionized Mg concentrations can be stored at 20°C for ≤24 hours, at 4°C for ≤72 hours, and at ?20°C for ≤4 weeks, when samples are minimally exposed to air. Intra‐ and interassay precisions had coefficients of variation (CVs) of 1.23% and 2.02%, respectively. There was good linearity using serum (r= .998; y=?0.0057 + 1.0256x) and manufacturer‐supplied aqueous solutions and quality control materials (r= .999; y= 0.0110 + 0.9213x). Apparent analytical sensitivity was at least 0.015 mmol/L. Mean recovery was good for ionized Mg in samples with ≤1+ icterus (104%), 4+ lipemia (99.3%) and 1–4+ hemolysis (98.6%). There was no significant difference (P= .52) in ionized Mg concentrations in serum collected in tubes containing no additives compared with serum collected in glass separator tubes. The serum ionized Mg reference interval was 0.47–0.63 mmol/L (n = 40). Conclusions: The Nova CRT8 analyzer and assay provide a precise and reliable method of measuring ionized Mg concentration in feline serum. Strict adherence to sampling techniques, handling, and storage are necessary for reliable results.     

Dietary phosphorus restriction affects bone metabolism,vitamin D metabolism and rumen fermentation traits in sheep

Homeostasis of calcium (Ca) and phosphate (P_i) is maintained by a concerted interplay of absorption and reabsorption via the gastrointestinal tract and the kidney and by storage and mobilization from the bone regulated mainly by parathyroid hormone (PTH), 1,25-dihydroxycholecalciferol and calcitonin. The present study aimed at characterizing the effects of dietary P restriction on bone, vitamin D metabolism and rumen fermentation traits reflecting the endogenous P cycle maintaining the ruminal P supply for microbial metabolism. The experiments were done in eleven female, non-pregnant, non-lactating four- to nine-year-old Black Headed Mutton sheep allotted to two feeding groups: "P-restricted" (0.11% P/kg DM and 0.88% Ca/kg DM) and "Control" (0.38% P/kg DM and 0.88% Ca/kg DM). Dietary P restriction did not lead to hypophosphataemia, probably due to a compensation by bone mobilization, demonstrated by increased serum concentrations of a resorption marker and altered gene expression in bone tissue. In addition, the RNA expression of fibroblast growth factor 23, a bone-derived factor involved in the regulation of vitamin D metabolism, was significantly reduced with dietary P restriction. Furthermore, several genes related to vitamin D metabolism and plasma concentrations of 1,25-(OH)₂D were associated with serum concentrations of phosphate (P_i). In the parotid gland, the expression of the P_i transporter NaPi2b was negatively associated with serum P_i and positively with parathyroid PTH expression. Although P_i concentrations in saliva and the gastrointestinal tract were significantly reduced, we found no adverse effects of the P-restricted ration on the production of short chain fatty acids, but slight differences in the production of butyrate as well as its relationship to rumen P_i and ammonia concentrations that might indicate an impact on ruminal fermentation.     

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca²⁺ ([Ca²⁺]i): transient elevations in [Ca²⁺]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca²⁺ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca²⁺]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca²⁺ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca²⁺]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca²⁺ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca²⁺]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca²⁺ wave and spiral-like Ca²⁺ oscillations, respectively.     

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

Rapid calcitonin response to experimental hypercalcemia in healthy horses

Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca²⁺) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5 mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca²⁺ concentrations increased from 6.2 ± 0.3 mg/dL to 9.9 ± 0.5 mg/dL (4 min; P < 0.01). Serum CT increased from 16.7 ± 8.0 pg/mL to 87.1 ± 55.8 pg/mL at 2 min, and 102.5 ± 51.1 pg/mL at 4 min (P < 0.01). Serum CT returned to baseline by 20 min, whereas serum Ca²⁺ returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca²⁺ or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca²⁺ concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.     

Growth hormone release induced by an amino acid mixture from primary cultured anterior pituitary cells of goats

The effects of amino acids on growth hormone (GH) release and cytosolic calcium concentration ([Ca²⁺]_i) were investigated in caprine anterior pituitary cells cultured for 3 d in Dulbecco modified Eagle medium. The addition of an amino acid mixture consisting of seven nonessential amino acids (NEAA: l-Asp, Gly, l-Ala, l-Ser, l-Pro, l-Asn, and l-Glu; concentration of each 12.5–200 μmol/l) in the medium significantly raised GH release from the cultured cells in a concentration-dependent manner with the maximum release at 200 μmol/l NEAA. Although an addition of l-Asp (0.1–100 μmol/l) caused a significant rise in GH release in a concentration-dependent manner, neither the individual amino acids contained in NEAA except l-Asp nor others (l-Leu, l-Phe, l-Gln, l-Met, and l-Arg) caused a rise in GH release when added alone to the medium. The rise in GH release induced by NEAA (200 μmol/l) and GH-releasing hormone (GHRH, 10 nmol/l) was significantly reduced by the addition of EGTA (l.8 mmol/l) and nifedipine (1 μmol/l) to the medium, respectively. The addition of NEAA (200 μmol/l) caused a rapid and transient [Ca²⁺]_i increase, followed thereafter by a steady increase. The prior addition of nifedipine (1 μmol/l), which itself significantly reduced the basal [Ca²⁺]_i, completely abolished the response induced by NEAA or GHRH. From these findings, we conclude that: 1) NEAA raises GH release and [Ca²⁺]_i in cultured caprine anterior pituitary cells, and 2) Ca²⁺ influx from the medium may be responsible for the cellular action of NEAA.