Circadian Variation of the Ca2+–PTH Curve during Hypercalcaemia in Dogs

To test the hypothesis that the parathyroid hormone (PTH) response to hypercalcaemia is influenced by circadian rhythms, the Ca²⁺–PTH curve was studied in six dogs after infusion of CaCl₂ (0.66 mEq/kg/h) at daytime (09:00 h) and at night-time (21:00 h). Plasma Ca²⁺ and PTH values measured before or after CaCl₂ infusion were not different at day and at night. However, in the recovery from hypercalcaemia, PTH concentrations were significantly lower (P < 0.05) at 21:00 h (23 ± 7.5 pg/ml at Ca²⁺ = 1.30 mm ) than at 09:00 h (38.8 ± 6.9 pg/ml at Ca²⁺ = 1.30 mm ). In addition, the Ca²⁺–PTH curve showed hysteresis at daytime (for the same Ca²⁺ concentration, PTH values were higher during recovery than during induction of hypercalcaemia) but not at night-time (PTH values were lower during recovery than during induction of hypercalcaemia). In conclusion, a circadian variation in the PTH secretory pattern during recovery from hypercalcaemia has been identified in dogs.     

Influence of dopexamine hydrochloride on haemodynamics and regulators of circulation in patients undergoing major abdominal surgery

Background: Catecholaminergic support is often used to improve haemodynamics in patients undergoing major abdominal surgery. Dopexamine is a synthetic vasoactive catecholamine with beneficial microcirculatory properties. Methods: The influence of perioperative administration of dopexamine on cardiorespiratory data and important regulators of macro- and microcirculation were studied in 30 patients undergoing Whipple pancreaticduodenectomy. The patients received randomized and blinded either 2 μg · kg^?1 · min^?1 of dopexamine (n=15) or placebo (n=15, control group). The infusion was started after induction of anaesthesia and continued until the morning of the first postoperative day. Endothelin-1 (ET-1), vasopressin, atrial natriuretic peptide (ANP), and catecholamine plasma levels were measured from arterial blood samples. Measurements were carried out after induction of anaesthesia, 2 h after onset of surgery, at the end of surgery, 2 h after surgery, and on the morning of the first postoperative day. Results: Cardiac index (CI) increased significantly in the dopexamine group (from 2.61±0.41 to 4.57±0.78 1 · min^?1 · m^?2) and remained elevated until the morning of the first postoperative day. Oxygen delivery index (DO₂I) and oxygen consumption index (VO₂I) were also significantly increased in the dopexamine group (DO₂I: from 416±91 to 717±110 ml/m² · m²; VO₂I: from 98±25 to 157±22 ml/m² · m²), being significantly higher than in the control group. pHi remained stable only in the dopexamine patients, indicating adequate splanchnic perfusion. Vasopressive regulators of circulation increased significantly only in the untreated control patients (vasopressin: from 4.37±1.1 to 35.9±12.1 pg/ml; ET-1: from 2.88±0.91 to 6.91±1.20 pg/ml). Conclusion: Patients undergoing major abdominal surgery may profit from prophylactic perioperative administration of dopexamine hydrochloride in the form of improved haemodynamics and oxygenation as well as beneficial influence on important regulators of organ blood flow.     

Haemodynamic,diuretic and hormonal responses to prostaglandin E1, infusion in halothane anaesthetized dogs: comparison among epidural lidocaine,epidural fentanyl and epidural saline

Deliberate hypotension decreases blood loss and transfusion but it may be accompanied by adverse effects due either to the hypotensive agents themselves or to haemodynamic alterations. Prostaglandin E₁ (PGE₁) has the advantage of a diuretic effect coupled with systemic hypotension. To elucidate the mechanisms by which PGE₁ induces diuresis we compared the haemodynamic, diuretic and hormonal responses to PGE₁ infusion simultaneously with epidural lidocaine (EP-L n = 7), epidural fentanyl (EP-F n = 8) or epidural saline (CONT n = 7) in halothane anaesthetized mongrel dogs. All groups developed a decrease in mean arterial pressure during PGE₁ infusion (from 105 ± 24 to 77 ± 18 mmHg in EP-L; 106 ± 19 to 79 ± 13 mmHg in the EP-F; and 129 ± 14 to 106 ± 18 mmHg in the CONT groups (mean ± SD)) (P < 0.05). In the EP-F and CONT groups urinary output increased during PGE₁ infusion (from 4.31 ± 1.89 to 6.15 ± 2.03 ml · min^?1 and 2.71 ± 1.23 to 4.48 ± 1.66 ml · min^?1 (P < 0.05), respectively) and was accompanied by increases in renal blood flow (from 87.0 ± 40.7 to 111.0 ± 42.8 ml · min^?1 and from 121.6 ± 46.6 to 158.4 ± 64.9 ml · min^?1 (P < 0.05), respectively) and in fractional excretion of sodium (FE_Na) (from 4.78 ± 3.88 to 7.63 ± 5.20% in CONT group). Plasma epinephrine concentration increased after laparotomy in the CONT group (from 0.09 ± 0.08 to 0.17 ± 0.14 pg · min^?1) (P < 0.05) and antidiuretic hormone (ADH) concentration increased after laparotomy (from 6.9 ± 5.2 to 21.0 ± 13.0 pg · ml^?1 in EP-F and from 8.1 ± 6.2 to 45.8 ± 29.9 pg · ml^?1 in CONT groups). Plasma renin activity increased after laparotomy in the EP-L group (from 2.00 ± 1.37 to 4.72 ± 2.73 mg · ml^?1 hr^?1) (P < 0.05). The results suggest that the mechansim of the PGE_1? induced diuretic effect includes increases in renal blood flow while renal sympathetic innervation is maintained and in FE_Na in the presence of elevated plasma ADH concentration.     

Intracellular free calcium and phosphatidyl inositol — 1,4,5 — triphosphate in bone cells cultured in a low calcium environment

The movement of intracellular free calcium([Ca²⁺]i) and phosphatidyl inositol-1, 4, 5-triphosphate (IP₃) was studied in bone cells cultured in a low-calcium environment. The [Ca²⁺]i was 98.0±10.2(n=6)nM/10⁶ cells for the control group (bone cells cultured in control medium) and 21.3±2.8(n=6)nM/10⁶ cells for the low Ca group (bone cells cultured in low Ca medium). After the addition of exogenous CaCl₂ to the calibration solution, [Ca²⁺]i increased significantly more in the low Ca group than in the control group(p<0.01). The IP₃ content/2×10⁶ cells was 12.40 pmoles in the control group and less than 0.19 pmoles in the low Ca group. After the stimulation with phospholipase C (PLC), the IP₃ content in the bone cells increased markedly more in the low Ca group than in the control group. These findings suggest that a low-calcium environment around cells and organsin vivo may inhibit the intracellular signal tranduction system.     

Comparison of metabolism of vitamins D2 and D3 in children with nutritional rickets

Children with calcium‐deficiency rickets may have increased vitamin D requirements and respond differently to vitamin D₂ and vitamin D₃. Our objective was to compare the metabolism of vitamins D₂ and D₃ in rachitic and control children. We administered an oral single dose of vitamin D₂ or D₃ of 1.25 mg to 49 Nigerian children—28 with active rickets and 21 healthy controls. The primary outcome measure was the incremental change in vitamin D metabolites. Baseline serum 25‐hydroxyvitamin D [25(OH)D] concentrations ranged from 7 to 24 and 15 to 34 ng/mL in rachitic and control children, respectively (p < .001), whereas baseline 1,25‐dihydroxyvitamin D [1,25(OH)₂D] values (mean ± SD) were 224 ± 72 and 121 ± 34 pg/mL, respectively (p < .001), and baseline 24,25‐dihydroxyvitamin D [24,25(OH)₂D] values were 1.13 ± 0.59 and 4.03 ± 1.33 ng/mL, respectively (p < .001). The peak increment in 25(OH)D was on day 3 and was similar with vitamins D₂ and D₃ in children with rickets (29 ± 17 and 25 ± 11 ng/mL, respectively) and in control children (33 ± 13 and 31 ± 16 ng/mL, respectively). 1,25(OH)₂D rose significantly (p < .001) and similarly (p = .18) on day 3 by 166 ± 80 and 209 ± 83 pg/mL after vitamin D₂ and D₃ administration, respectively, in children with rickets. By contrast, control children had no significant increase in 1,25(OH)₂D (19 ± 28 and 16 ± 38 pg/mL after vitamin D₂ and D₃ administration, respectively). We conclude that in the short term, vitamins D₂ and D₃ similarly increase serum 25(OH)D concentrations in rachitic and healthy children. A marked increase in 1,25(OH)₂D in response to vitamin D distinguishes children with putative dietary calcium‐deficiency rickets from healthy children, consistent with increased vitamin D requirements in children with calcium‐deficiency rickets. © 2010 American Society for Bone and Mineral Research     

Inhibition of CaMKII Attenuates Progressing Disruption of Ca2+ Homeostasis Upon Left Ventricular Assist Device Implantation in Human Heart Failure

In heart failure, left ventricular assist device (LVAD) implantation is performed to ensure sufficient cardiac output. Whereas some patients are subsequently weaned from LVAD support, other patients still need heart transplantation. To elucidate underlying mechanisms, we assessed the arrhythmogenic SR‐Ca²⁺ leak at the time of LVAD implantation (HF‐Im) and heart transplantation (HF‐Tx) and evaluated the effects of CaMKII‐inhibition. Human left‐ventricular cardiomyocytes were isolated, paced at 1 Hz for 10 beats to ensure SR‐Ca²⁺ loading and scanned for diastolic Ca²⁺ sparks (confocal microscopy). In HF‐Im, the high diastolic spark frequency (CaSpF) of 0.76 ± 0.12 × 100 μm^?1 × s^?1 could be reduced to 0.48 ± 0.10 × 100 μm^?1 × s^?1 by CaMKII inhibition (AIP, 1 μM). The amplitude of Ca²⁺ sparks, width, and length was not significantly altered. In sum, CaMKII inhibition yielded a clear tendency toward a reduction of the SR‐Ca²⁺ leak (n cells/patients = 76/6 vs. 108/6, P = 0.08). In HF‐Tx, we detected an even higher CaSpF of 1.00 ± 0.10 100 μm^?1 × s^?1 and a higher SR‐Ca²⁺ leak compared with HF‐Im (increase by 81 ± 33%, n cells/patients = 156/7 vs. 130/7, P < 0.05), which fits to the further decreased LV function. Here, CaMKII inhibition likewise reduced CaSpF (0.35 ± 0.09 100 μm^?1 × s^?1, P = 0.06) and significantly reduced spark duration (n sparks/patients = 58/3 vs. 159/3, P < 0.05). Conclusively, the SR‐Ca²⁺ leak was reduced by 69 ± 12% in HF‐Tx upon CaMKII inhibition (n cells/patients = 53/3 vs. 91/3, P < 0.05). These data show that the SR‐Ca²⁺ leak correlates with the development of LV function after LVAD implantation and may represent an important pathomechanism. The fact that CaMKII inhibition reduces the SR‐Ca²⁺ leak in HF‐Tx suggests that CaMKII inhibition may be a promising option to beneficially influence clinical course after LVAD implantation.     

Calcitriol pulse therapy is not more effective than daily calcitriol therapy in controlling secondary hyperparathyroidism in children with chronic renal failure

Calcitriol oral pulse therapy has been suggested as the treatment of choice for secondary hyperparathyroidism, but its efficacy and safety are still under discussion. The present randomized multicenter study compares the effect of an 8-week course of daily versus intermittent (twice weekly) calcitriol therapy on parathyroid hormone (PTH) suppression in 59 children (mean age 8.4±4.7 years) with chronic renal insufficiency (mean C_cr 22.4±11.6 ml/min per 1.73 m²) and secondary hyperparathyroidism. After a 3-week washout period, the patients were randomly assigned to treatment with daily oral calcitriol (10 ng/kg per day) or intermittent oral calcitriol (35 ng/kg given twice a week). The calcitriol dose was not changed throughout the study period of 8 weeks. At start of the study, the median intact PTH (iPTH) level was 485 pg/ml (range 83–2032) in the daily group (n=29) and 315 pg/ml (range 93–1638) in the intermittent group (n=30). After 8 weeks, the respective median iPTH concentrations were 232 pg/ml (range 63–1614) and 218 pg/ml (range 2–1785) (ns). The mean iPTH decrease from baseline was 19.2±57.8% and 13.7±46.7% respectively (not significant). Calcitriol reduced the iPTH concentration in 23/29 patients in the daily group and in 21/30 in the intermittent group. One episode of hypercalcemia (>11.5 mg/dl) was observed in both groups and a single episode of hyperphosphatemia (>7.5 mg/dl) was observed in the daily group. It is concluded that oral calcitriol pulse therapy does not control secondary hyperparathyroidism more effectively than the daily administration of calcitriol in children with chronic renal failure prior to dialysis. Received: 29 September 1999 / Revised: 2 February 2000 / Accepted: 9 February 2000     

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules

Calcium(Ca²⁺)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of <3-week-old (newborn) and >12-week-old (adult) rabbits. Tubules were loaded with 10 μM fura-2 AM by incubation for 60 min at 37°C, and then superfused with isosmotic saline solution gassed with either 95%O₂-5%CO₂ (control group) or 95%N₂-5%CO₂ (anoxia group) for 30 min. [Ca²⁺]_i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca²⁺]_i in adult tubules (from resting values of 245±10 to 975±100 nM, P <0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137±5 to 165±5 nM, P <0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca²⁺]_i from baseline, to 920±90 nM in tubules from adult and to 396±16 nM in those from newborn rabbits (P <0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca²⁺]_i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca²⁺]_i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (¹⁴C-α-methyl-glucopyranoside) and phosphate (³²Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca²⁺ rises to critical levels in PCTs of adults compared with those of <3-week-old animals, (2) Ca²⁺ influx occurs via a pathway activated by exposure to high [K⁺]_o, presumably voltage-sensitive Ca²⁺ channels or reversal of Na⁺-Ca²⁺ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits. Received June 27, 1995; received in revised form December 1, 1995; accepted December 8, 1995     

Alteration in osteoblast activity and nutritional vitamin-D deficiency in non-hypercalcemic malignancy

Summary The biochemical parameters of bone mineral metabolism in patients with nonhypercalcemic malignancy have not been extensively investigated. Therefore, a group of 29 such patients with different types of malignancy was studied. Ten patients received corticosteroids. In the entire group, serum ionized calcium (Ca²⁺), bone gla protein (BGP), 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D (1,25(OH)₂D) were all lower than in age-matched controls, and carboxy-terminal parathyroid hormone (CPTH) was higher. Although both corticosteroid- and noncorticosteroid-treated patients had decreased BGP values, the corticosteroid-treated patients had lower BGP levels than those not on steroids (4.24±0.70 SE vs. 11.50±2.20 ng/ml;P<0.005). Patients on corticosteroids had lower 1,25(OH)₂D values than controls (18.81 ±2.71 vs. 27.83±1.17 pg/ml;P<0.01), whereas those not on corticosteroids had normal 1,25(OH)₂D values. These results suggest that patients with nonhydpercalcemic malignancy have nutritional vitamin-D deficiency and secondary hyperparathyroidism with perhaps corticosteroid-induced suppression of serum 1,25(OH)₂D and BGP. The decreased levels of serum BGP in the nonsteroid-treated patients suggest, in addition, a defect in osteoblast function.     

Rat models of normocalcemic hypercalciuria of different pathogenic mechanisms

Hypercalciuria was induced in female Sprague-Dawley rats, aged 40±2 days, by 7-day administration (mean±SEM) of calcitriol (5.4±0.1 ng/100 g per day, intraperitoneal), furosemide (14.9±1.9 mg/100 g per day, oral), or ammonium chloride (3.8±0.1 mEq/100 g per day, oral). Calciuria increased from 1.9±0.2, 1.6±0.2, and 1.9±0.3 to 5.4±0.5, 4.0±0.9, and 5.4±0.5 mg/100 g per day in the calcitriol (VD, n = 9), furosemide (F, n = 6), and ammonium chloride (AC, n = 10) groups, respectively. Calciuria did not change (1.9±0.3 vs. 1.6±0.1 mg/100 g per day) in control rats (n = 8). Ninety-six percent of treated rats became hypercalciuric as assessed by urine calcium excretion above the 90th percentile of normal values. Hypercalciuria was of similar degree in the three groups of rats and was not associated with hypercalcemia, metabolic acidosis, severe serum electrolyte imbalance, or growth impairment. VD rats had low serum parathyroid hormone (PTH) concentrations (3.0±0.5 pg/ml vs. 15.8±1.3 pg/ml in controls, P <0.05), whereas serum PTH was not significantly elevated in F rats (16.2±1.8 pg/ml). Thus, the protocol caused three forms of hypercalciuria that mimicked the clinical conditions of idiopathic hypercalciuria in humans and may clearly be differentiated according to their mechanism of production. This experimental model of normocalcemic hypercalciuria may be useful to clarify unknown aspects of pathogenesis and pathophysiology of idiopathic hypercalciuria in children. Received March 12, 1997; received in revised form September 19, 1997; accepted September 24, 1997     

Effect of Chronic Vitamin D Infusion Upon In Vivo Glucose Disposal

We have previously demonstrated that parathyroid hormone (PTH) infusion decreases glucose disappearance rate (K_g) in vivo. Because in the rodent model used it was not possible to determine whether the PTH itself, the induced hypercalcemia, or both contributed to the glucose intolerance, we examined the effect of vitamin D infusion on insulin-mediated glucose disposal. In this model also hypercalcemia is induced but PTH levels are suppressed. Thirty male Sprague Dawley rats were continuously infused with vit D for 5 days using an Alzet miniosmotic pump, at a rate of 9.7 pmol/hour. Thirty controls were infused with the vehicle alone. On the 5th day, glucose 700 mg/kg and insulin 0.35 U/kg were given as a bolus through the left femoral vein and blood samples were obtained from the right femoral vein just prior to and at 2, 5, 10, and 20 minutes post-glucose/insulin infusion. At the end of 5 days, plasma calcium levels were higher in the vit D-infused rats than in the control rats (12.8 ± 0.1 versus 10.0 ± 0.1 mg/dL, P < 0.01) and rat PTH levels were suppressed (2.1 ± 0.1 versus 62 ± 12 pg/ml, P < 0.01). Glucose levels were higher in the vit D animals only at 5 minutes following glucose/insulin bolus (375 ± 7 versus 350 ± 6 mg/dL, P < 0.01) but at no other time. There were no differences between serum insulin levels at any time. Unlike previous findings in PTH-infused rats, K_g (measured from 2 to 20 minutes following glucose/insulin bolus) was not different between groups (4.5 ± 0.3 versus 4.7 ± 0.2, P= 0.92.) A positive correlation between serum calcium and serum glucose was found only at 5 minutes (r = 0.55, P < 0.01) and only in the vit D animals. The areas under the glucose curves approached statistically significant differences (vit D-infused 5258 ± 142 mg/dL/18 minutes versus control 4947 ± 127, P= 0.06.) Analysis of serum glucose data by two-factor analysis of variance (ANOVA) suggests that the two groups differ slightly in glucose values (P= 0.03) but have parallel K_g. In order to define whether different effects of PTH (1–34) and vit D on intracellular calcium [Ca²⁺]_i levels could partly explain the different effects of PTH and vit D infusion on glucose disposal, we investigated the effect of PTH and vit D infusions on basal and concanavalin A (con A)-stimulated changes in mononuclear [Ca⁺²]_i levels. Following 5 days of PTH, vit D, or control infusion, peripheral mononuclear cells were incubated with 50 μg/ml con A. Changes in [Ca⁺²]_i over 5 minutes were calculated by flow cytometric measurement of the calcium sensitive fluo-3 AM dye. Despite achieving significant and comparable degrees of hypercalcemia in the PTH and vit D infused animals, there were no differences in basal or con A-stimulated [Ca⁺²]_i levels from control. Consequently, we conclude that vit D-induced hypercalcemia associated with suppressed PTH levels has mild affects on glucose homeostasis but does not affect glucose disappearance rate in vivo (K_g) as does hypercalcemia induced by PTH infusion, and that neither chronic PTH infusion nor chronic vit D infusion are associated with long-standing changes in [Ca²⁺]_i levels. Received: 24 March 1998 / Accepted: 29 June 1998     

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: Regulation by 1,25-dihydroxycholecalciferol

Summary To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD₃, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] on³H-25OHD₃ were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of³H-25OHD₃, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)₂D₃ (50 ng/day i.p. for 7 days) increased mean serum³H-24,25-dihydroxycholecalciferol [³H-24,25(OH)₂D₃] from 2±2–210±80 fmol/ml (mean±1 SD), increased mean serum³H-25,26-dihydroxycholecalciferol [³H-25,26(OH)₂D₃] from 2±2–12±6 fmol/ml and lowered mean serum³H-1,25(OH)₂D₃ from 210±40–4±4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)₂D₃ increased mean serum³H-24,25-(OH)₂D₃ from 6±11–115±30 fmol/ml and increased mean serum³H-25,26(OH)₂D₃ from 3±3–26 ± 10 fmol/ml. Nephrectomy increased serum³H-25(OH)D₃ in untreated (from 1450±225–2675±225 fmol/ml serum) and 1,25(OH)₂D₃ treated rats (from 1600±175–3075±100 fmol/ml).³H-1,25(OH)₂D₃ averaged 74±16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)₂D₃ production and for most 25,26(OH)₂D₃ production. Further, the observed stimulation of production of 24,25(OH)₂D₃ and 25,26(OH)₂D₃ by 1,25(OH)₂D₃ in anephric — D rats providesin vivo evidence for regulation of extrarenal 25OHD₃: 24- and 26-hydroxylases.     

Effects of Lead, Mercury, and Methyl Mercury on Gap Junctions and [Ca2+]i in Bone Cells

Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca²⁺]_i). Since heavy metals compete with Ca²⁺ at multiple sites and increased [Ca²⁺]_i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca²⁺]_i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb_(e) (5 μmol/liter; n = 3) and Hg_(e) (5 μmol/liter; n = 3) as well as Pb_(i) (25 μmol/liter; n = 7) did not change the coupling (ΔMP₂/ΔMP₁). In contrast, MeHg_(e) (1–10 μmol/liter; n = 6) and Hg_(i) (≥5 μmol/liter; n = 8) reduced the coupling to 79.5 ± 19.3% and 62.4 ± 15.3%, respectively, within 15–20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb_(e) (5 μmol/liter, n = 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca²⁺]_i increase. Hg_(e) (n = 48) slightly increased [Ca²⁺]_i from 100 to ≤200 nmol/liter, whereas MeHg_(e) (5 μmol/liter, n = 52) released Ca²⁺ from internal stores, thus increasing [Ca²⁺]_i up to 2 μmol/liter. In conclusion, Pb_(e), Pb_(i) and Hg_(e) do not affect gap junctional coupling per se. Since MeHg_(e) and Hg_(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca²⁺]_i, which affects gap junction channels. Received: 6 May 1997 / Accepted: 15 October 1997     

Increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage in the cultured neurons of Lymnaea stagnalis

Purpose To determine whether the increase in intracellular Ca²⁺ concentration induced by lidocaine produces neurotoxicity, we compared morphological changes and Ca²⁺ concentrations, using fura-2 imaging, in the cultured neurons of Lymnaea stagnalis. Methods We used BAPTA-AM, a Ca²⁺ chelator, to prevent the increase in the intracellular Ca²⁺ concentration, and Calcimycin A23187, a Ca²⁺ ionophore, to identify the relationship between increased intracellular Ca²⁺ concentrations and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells. Results Increasing the dose of lidocaine increased the intracellular Ca²⁺ concentration; however, there was no morphological damage to the cells in lidocaine at 3 × 10⁻³ M. Lidocaine at 3 × 10⁻² M increased the intracellular Ca²⁺ concentration in both saline (from 238 ± 63 to 1038 ± 156 nM) and Ca²⁺-free medium (from 211 ± 97 to 1046 ± 169 nM) and produced morphological damage and shrinkage, with the formation of a rugged surface. With the addition of BAPTA-AM, lidocaine at 3 × 10⁻² M moderately increased the intracellular Ca²⁺ concentration (from 150 ± 97 to 428 ± 246 nM) and produced morphological damage. These morphologically changed cells were stained dark blue with trypan blue dye. The Ca²⁺ ionophore increased the intracellular Ca²⁺ concentration (from 277 ± 191 to 1323 ± 67 nM) and decreased it to 186 ± 109 nM at 60 min. Morphological damage was not observed during the 60 min, but became apparent a few hours later. Conclusion These results indicated that the increase in intracellular Ca²⁺ concentration is not the only cause of lidocaine-induced cell damage.     

Tissue specificity and mechanism of vitamin D receptor up-regulation during dietary phosphorus restriction in the rat

Dietary phosphorus restriction up-regulates intestinal vitamin D receptor (VDR), but the tissue specificity of the up-regulation and the mechanism of receptor accumulation remain unknown. Therefore, the effects of low phosphorus diet (LPD) on VDR content in intestine, kidney, and splenic monocytes/macrophages were examined. Male Sprague-Dawley rats weighing 50–100 g were fed a normal diet (NPD; 0.6% Ca, 0.65% P) as controls followed by an LPD (0.6% Ca, 0.1% P) for 1–10 days (D1-D10). LPD rapidly decreased serum P levels by D1 from 11.11 ± 0.19 mg/dl (mean ± SE) to 4.98 ± 0.37 mg/dl (n = 9). LPD increased total serum Ca from 10.54 ± 0.09 mg/dl to 11.63 ± 0.15, 12.17 ± 0.15, and 12.39 ± 0.18 mg/dl by D1, D2, and D3, respectively, and then remained stable. Serum 1,25-(OH)₂D₃ rapidly increased from 123 ± 5.4 pg/ml to 304 ± 35 pg/ml by D1, reached a plateau through D5, and then gradually increased to 464.9 ± 27.7 pg/ml by D10. Intestinal VDR quantitated by ligand binding assay increased 3.5-fold from 169.6 ± 13.7 fmol/mg of cytosol protein in rats fed NPD (n = 12) to a peak of 588.3 ± 141.88 fmol/mg of protein by D3 (n = 6; p < 0.001) and then decreased to a plateau level of 2.5-fold greater than NPD (p < 0.05) during D5 to D10. In contrast, LPD did not up-regulate kidney or splenic monocyte/macrophage VDR. Northern blot analysis showed that intestinal VDR mRNA increased 2-fold by D2 (n = 3) of LPD and then gradually decreased to control levels after D5. In contrast, kidney VDR mRNA levels did not change during the first 5 days of P restriction and then subsequently decreased to 50% of NPD controls. The results of these studies indicate that VDR up-regulation during dietary phosphorus restriction is tissue-specific and that the mechanism of the up-regulation is time-dependent. Acutely (D1-D5), phosphorus restriction up-regulates intestinal VDR through increased VDR gene expression, whereas chronic (D5-D10) phosphorus restriction appears to alter VDR metabolism through nongenomic mechanisms that are consistent with prolongation of the half-life of the receptor. The nature of the tissue-specific regulation of VDR during phosphorus restriction remains to be determined.     

Effect of maternal insulin deficiency on vitamin D metabolite concentrations in normoglycemic pregnant rats and their fetuses

Summary The effects on vitamin D metabolite concentrations of insulin deficiency, not accompanied by hyperglycemia, were investigated in pregnant rats and in their fetuses injected with 75 mg/kg BW streptozotocin (SZ). These concentrations were measured in maternal plasma and whole fetal body. In the insulinopenic mothers, the 25OHD concentration was found to rise compared with that of control pregnant rats (7.00±1.66 ng/ml, n=16, versus control 4.50±1.60, n=10, 0.001<P<0.01). The concentration of 1,25 (OH)₂D, which was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was not different in our control and insulinopenic rats (107.36±38.25 pg/ml, n=11, versus control 122.90±18.20, n=8). In fetuses from our SZ-injected rats, the 24,25 (OH)₂D level diminished compared with the control level (2.12±0.70 ng/g, n=11, versus control 5.23±0.95 ng/g, n=13,P<0.001). The Ca/P ratio in fetal body also decreased (0.68 versus control 1.12). It is suggested that the placental metabolism is an important determinant of normal fetal growth.     

Nicotine inhibits large conductance Ca2 + -activated K+ channels and the NO/-cGMP signaling pathway in cultured human endothelial cells

Objective. The effects of nicotine on endothelium-dependent vasorelaxation mediated by nitric oxide (NO) are controversial. Since endothelial NO synthesis has been shown to depend on the activity of large conductance Ca^2?+?-activated K⁺ channels (BK_Ca), the present study investigated whether nicotine alters BK_Ca single channel activity induced by the K⁺ channel opener NS1619, and to examine a possible interaction with the endothelial NO generation. Design. The patch-clamp technique was used to examine the BK_Ca activity. NO production was measured indirectly using a [³H]-cGMP-radioimmunoassay. All experiments were performed using cultured endothelial cells derived from human umbilical cord veins. Results.The BK_Ca opener NS1619 (10 µmol/l) significantly increased the BK_Ca open-state probability (NPo) from 0.011±0.007 (control) to 0.052±0.019. Co-perfusion with nicotine (1 µmol/l) significantly decreased NS1619 induced NPo (n?=?14, p?<?0.05). Intracellular cGMP levels were significantly increased, if cells were stimulated with NS1619 (+ 225%; n?=?10, p?<?0.05), which was blocked by Nicotine (1 µmol/l). Conclusions. The results of the present study demonstrate that BK_Ca activation by NS1619 plays an important role in the regulation of the NO-/cGMP-signaling-pathway. Endothelial dysfunction caused by nicotine may be connected with a decrease in BK_Ca-activity.     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Isoflurane and halothane impair both systolic and diastolic function in the newborn pig

Purpose

Volatile anaesthetics have considerable effects on diastolic relaxation in the adult myocardium. We hypothesized that isoflurane (I) and halothane (H) may have even greater effects on diastolic function in the newborn, as the newborn heart has increased passive stiffness and altered calcium handling relative to the adult. Using a newborn pig model, we compared I and H at three clinically relevant concentrations with respect to both systolic and diastolic function.

Methods

Sixteen newborn pigs were randomized for study at control (background fentanyl 100 μg · kg^?1 · hr^?1 and 0.5, 1.0 and 1.5 MAC of I (n = 8) or H (n = 8). Temperature, arterial blood gasex, and LVEDP were controlled. Left ventricular pressure (LVP) was monitored continuously and LV anteriorposterior dimension was determined by using sonomicrometry crystals. Systolic function was assessed by peak positive dP/dT (dP/dT_max) and the slope of the end-systolic pressure-dimension (ESP-D) relationship. Diastolic relaxation was given by peak negative dP/dT (?dP/dT_max) and the time constant for ventricular relaxation (τ). Left ventricular stiffness was calculated from the slope of the end-diastolic pressure-dimension (EDP-D) relationship.

Results

At equal MAC concentrations, I and H were identical in effect for every variable studied. Systolic function was depressed at all anaesthetic concentrations. Control dP/dT_max (1:1815 ± 561 (SD) mmHg · sec^?1, H:1841 ± 509) decreased to 832 ± 341 with 1.5 MAC I and 691 ± 127 with 1.5 MAC H (P < 0.05 vs control). ESP-D slope decreased from 62 ± 31 mmHg · mm^?1 at control to 15 ± 11 with 1.5 MAC I and from 79 ± 16 to 37 ± 15 with 1.5 MAC H (P < 0.05 vs control). Diastolic function was affected only at higher MAC anaesthesia. Control τ increased from 18.0 ± 6 msec to 29.1 ± 7.5 with 1.5 MAC I and from 20.8 ± 5.9 to 30.0 ± 11.3 with 1.5 MAC H (P < 0.05). EDP-D slope was increased at both 1 and 1.5 MAC anaesthesia. EDP-D slope increased from 0.16 ± 0.24 mmHg · mm^?1 at control to 0.58 ± 0.46 with 1 MAC I and from 0.16 ± 11 to 0.50 ± 0.35 with 1 MAC H. The ?dP/dT_max decreased at every MAC level of anaesthesia.

Conclusion

These combined systolic and diastolic effects help to explain the increased sensitivity of the newborn myocardium to volatile anaesthetics.     

Effects of hPTH(1‐34) Infusion on Circulating Serum Phosphate, 1,25‐Dihydroxyvitamin D,and FGF23 Levels in Healthy Men

Fibroblast growth factor 23 (FGF23) promotes phosphaturia and suppresses 1,25‐dihydroxyvitamin D [1,25(OH)₂D] production. PTH also promotes phosphaturia, but, in contrast, stimulates 1,25(OH)₂D production. The relationship between FGF23 and PTH is unclear, and the acute effect of pharmacologically dosed PTH on FGF23 secretion is unknown. Twenty healthy men were infused with human PTH(1‐34) [hPTH(1‐34)] at 44 ng/kg/h for 24 h. Compared with baseline, FGF23, 1,25(OH)₂D, ionized calcium (iCa), and serum N‐telopeptide (NTX) increased significantly over the 18‐h hPTH(1‐34) infusion (p < 0.0001), whereas serum phosphate (PO₄) transiently increased and then returned to baseline. FGF23 increased from 35 ± 10 pg/ml at baseline to 53 ± 20 pg/ml at 18 h (p = 0.0002); 1,25(OH)₂D increased from 36 ± 16 pg/ml at baseline to 80 ± 33 pg/ml at 18 h (p < 0.0001); iCa increased from 1.23 ± 0.03 mM at baseline to 1.46 ± 0.05 mM at hour 18 (p < 0.0001); and NTX increased from 17 ± 4 nM BCE at baseline to 28 ± 8 nM BCE at peak (p < 0.0001). PO₄ was 3.3 ± 0.6 mg/dl at baseline, transiently rose to 3.7 ± 0.4 mg/dl at hour 6 (p = 0.016), and then returned to 3.4 ± 0.5 mg/dl at hour 12 (p = 0.651). hPTH(1‐34) infusion increases endogenous 1,25(OH)₂D and FGF23 within 18 h in healthy men. Whereas it is possible that the rise in PO₄ contributed to the observed increase in FGF23, the increase in 1,25(OH)₂D was more substantial and longer sustained than the change in serum phosphate. Given prior data that suggest that neither PTH nor calcium stimulate FGF23 secretion, these data support the assertion that 1,25(OH)₂D is a potent physiologic stimulator of FGF23 secretion.