EGTA Infusion Research Articles

Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7g), lactose (5.1g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na+ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K+ concentrations decreased. As involution advanced, the Na+:K+ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na+ concentration and in the Na+:K+ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.

Characterization of Basal and Morphine-Induced Uridine Release in the Striatum: An In Vivo Microdialysis Study in Mice

Uridine, a pyrimidine nucleoside, has been proposed to be a potential signaling molecule in the central nervous system. The understanding of uridine release in the brain is therefore of fundamental importance. The present study was performed to determine the characteristics of basal and morphine-induced uridine release in the striatum of freely moving mice by using the microdialysis technique. To ascertain whether extracellular uridine was derived from neuronal release, the following criteria were applied: sensitivity to (a) K(+) depolarization, (b) Na(+) channel blockade and (c) removal of extracellular Ca(2+). Uridine levels were not greatly affected by infusion of tetrodotoxin (TTX) and were unaffected by either Ca(2+)-free medium or in the presence of EGTA (a calcium chelator), suggesting that basal extracellular uridine levels were maintained mainly by non-vesicular release mechanisms. In addition, both systemic and local application of morphine increased striatal uridine release. The morphine-induced release was reversed by naloxone pretreatment, but was unaffected by TTX or EGTA infusion. Moreover, co-administration of morphine and nitrobenzylthioinosine (NBTI, an inhibitor of nucleotide transporter) produced increases of uridine levels similar to that produced by NBTI or morphine alone, suggesting a nucleotide transporter mechanism involved. Taken together, these findings suggest that morphine produces a μ-opioid receptor-mediated uridine release via nucleoside transporters in a TTX- and calcium-independent manner.

Disruption of cell junctions induces apoptosis and reduces synthetic activity in lactating goat mammary gland

Although it is known that disruption of the cell junctions in the mammary gland induces a decrease in milk yield, the cellular mechanisms involved in milk secretion reduction during mammary cell junction disruption are not well understood. The aim of this study was to investigate the cellular regulations taking place after cell junction disruption in the mammary gland of goats. We performed intramammary infusions of Ca chelators to induce cell junction disruption. In a first group of 5 goats, intramammary infusions of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) in the right gland halves and saline as a control in the left gland halves were performed after 4 consecutive milkings. A second group of 4 goats received 4 intramammary infusions of citrate solution in the right gland halves and lactose solution as a control in the left halves. Intramammary infusion of EGTA and lactose induced a disruption of cell junctions, whereas citrate infusions failed to modify mammary epithelium integrity. The effect of the infused solutions was also tested in vitro via the measurement of transepithelial resistance, confirming mammary epithelium disruption by the EGTA, lactose, and citrate solutions at high concentrations. The disruption of mammary epithelium integrity by EGTA induced a decrease in the expression of the cell junction protein E-cadherin. Both the EGTA and lactose infusions induced a decrease in milk secretion that was accompanied by cellular modifications. We observed a decrease in milk casein, which was associated with a decrease in the mRNA level of κ-casein in the lactose-infused glands, and a decrease in milk lactose, which was associated with a downregulation of α-lactalbumin transcripts in both the EGTA- and lactose-treated glands. Both the EGTA and lactose infusions increased terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) in the mammary tissue, indicating an induction of apoptosis. Lactose infusion increased the mRNA level of Bax, suggesting that apoptosis was regulated at the transcriptional level. The results obtained in these experiments suggest that disruption of mammary epithelium integrity was associated with both reduced synthetic activity and apoptosis induction in the mammary gland.

Calcium‐sensing receptor and recovery from hypocalcaemia in thyroparathyroidectomized rats

Plasma ionized calcium (p-Ca(2+)) is kept within a very narrow range and deviations are rapidly corrected by flux of Ca(2+) between extracellular fluid and the labile calcium pool at the quiescent bone surface. The calcium sensing at the bone surface represents a physiological interesting model for the rapid minute-to-minute regulation of p-Ca(2+). Our aim was to study whether the calcium-sensing receptor (CaR) has a role in the rapid recovery of p-Ca(2+) from acute induced hypocalcaemia. Male Wistar rats were thyroparathyroidectomized (TPTX). Acute hypocalcaemia in the animals was induced by infusion of EGTA (40-50 mM EGTA, 3.0 mL h(-1) for 30 min). Thereafter the recovery of p-Ca(2+) was followed. Vehicle or the CaR activators, R-568 (2 mg as a bolus twice) or gentamycin were administrated intravenously. EGTA infusion resulted in significantly lower nadir of hypocalcaemia in R-568- or gentamycin-treated rats compared to vehicle-treated rats (P < 0.01). During recovery phase p-Ca(2+) remained significantly lower in R-568 rats (P < 0.001). As such p-Ca(2+) levels recovered to basal levels in the vehicle group within 70 min after stopping EGTA, while R-568 or gentamycin rats remained significantly hypocalcaemic. The CaR activators R-568 and gentamycin, both significantly delayed the recovery of p-Ca(2+) from acute EGTA-induced hypocalcaemia in TPTX rats. This novel finding suggests the existence of calcium sensing by bone of importance for the rapid minute-to-minute regulation of p-Ca(2+).

Role of Ca 2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30–300 pmol), PNV (1–10 μg), Bay K-8644 (0.3–3 nmol) and KCl (10–100 μmol) dose-dependently contracted the preparations. Ca 2+-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 μM) and verapamil (10 μM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75–90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 μM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca 2+ from extracellular sources.

Rate-dependency of calcitonin secretion in response to increased plasma Ca2+.

Calcitonin (CT) is a polypeptide hormone secreted from C-cells of the thyroid gland in response to hypercalcemia. The physiological contribution of CT to calcium homeostasis has not been completely clarified. The present study therefore further characterized the sigmoidal relationship between plasma ionized calcium (P-Ca2+) and CT in normal rats, and examined the possibility of rate-dependency of CT secretion in response to changes in P-Ca2+. Hypercalcaemia was induced by an infusion of calcium gluconate at rate of 4.5 x 10(-2) mmol h-1 rat-1 i.v. (n = 8) and hypocalcaemia was induced by an EGTA infusion at a rate of 4.5 x 10(-2) mmol h-1 rat-1 (n = 7) in one protocol: the 'slow' protocol. In another protocol an increased rate of infusion of calcium gluconate or EGTA was used to induce a more rapid change in P-Ca2+. Calcium gluconate was infused at a rate of 6.0 x 10(-2) mmol h-1 rat-1 (n = 6) and EGTA infused at a rate of 7.5 x 10(-2) mmol h-1 rat-1 (n = 7): the 'rapid' protocol. The infusions of both the 'slow' and 'rapid' protocols resulted in linear changes in P-Ca2+, but with significantly different slopes (P < 0.01). The Ca2+/CT curves of both protocols were represented by sigmoidal curves. The 'rapid' increase of P-Ca2+ resulted in a higher maximal CT secretion (2032 +/- 215 pg mL-1) than the 'slow' increase of P-Ca2+ (1213 +/- 85 pg mL-1; P < 0.001), despite similar minimal and maximal levels being obtained in P-Ca2+ in the two protocols. Thus, a significantly greater CT response was obtained with a more rapid increment in P-Ca2+. The relationship between P-Ca2+ and CT is represented by a sigmoidal curve, as previously shown. The CT response depended, however, not only upon the concentration of P-Ca2+ obtained but also upon the rate of increase in P-Ca2+, demonstrating rate-dependency as another significant physiological relation between Ca2+ and CT.

Role of calcitonin in the rapid minute-to-minute regulation of plasma Ca2+ homeostasis in the rat.

Plasma ionized calcium (Ca2+) is maintained at a very stable concentration in mammals. The hormones or factors involved in the very rapid regulation of calcium homeostasis are still debated. Thus, previous results from our laboratory have clearly shown that parathyroid hormone (PTH) and 1,25(OH)2D3 are not responsible for the rapid up-regulation of plasma Ca2+ after a brief induction of hypocalcaemia. The present investigation therefore examined in vivo the possible role of calcitonin (CT) in the very rapid, minute-to-minute regulation of plasma Ca2+ in rats. The rapid calcaemic response to acute thyroparathyroidectomy (TPTX) and to acute selective thyroidectomy (TX) (n = 10), as well as the possible effect of CT on the very rapid recovery of plasma Ca2+ after termination of a brief induction of hypocalcaemia were studied. Hypocalcaemia was induced by a 30-min EGTA infusion in ras in three different protocols: 1 h after TPTX (n = 9) compared with control TPTX rats not given EGTA (n = 13); 1 h after TX (n = 7); and 1 h after TPTX, but during supplementation with exogenous CT (n = 8) and compared with the response in TPTX rats infused with vehicle (n = 8). An immediate and significant increase of plasma Ca2+ was found after TPTX (P < 0.01) as well as after selective TX (P < 0.01) in the nonfasting rats. Significant hypercalcaemia (P < 0.05) was still present in rats fasting for 2 days before these procedures, but the increase in plasma Ca2+ was considerably less (P < 0.01). After induction of a brief period of hypocalcaemia by infusion of EGTA a significant (P < 0.01) and rapid recovery of plasma Ca2+ took place within 10 min and a further increase within the next 60 min (P < 0.01), whether or not the rats were normal, TPTX, TX or were supplemented by CT during the experiments. The plasma Ca2+ recovery curves after termination of a brief induction of hypocalcaemia all had similar appearances, indicating that presence or absence of CT had no influence on this very rapid Ca2+ recovery after induction of hypocalcaemia. Acute removal of the tonus of CT results in an acute increase in plasma Ca2+ for up to 3 h. This effect of CT is probably mainly related to the postprandial maintenance of normocalcaemia, but is also seen in fasting rats, although to a lesser degree. The very rapid calcaemic recovery after discontinuation of a brief induction of hypocalcaemia is, however, not a result of suppressed plasma calcitonin levels.

Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells.

Persistence of capacitative Ca(2+) influx in inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-deficient DT40 cells (DT40(IP(3)R-/-)) raises the question of whether gating of Ca(2+)-release activated Ca(2+) current (I(crac)) by conformational coupling to Ca(2+)-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of I(crac) Ca(2+) current in wild-type and DT40(IP(3)R-/-) cells. In both cell types passive depletion of internal Ca(2+) stores by infusion of EGTA activated a Ca(2+) current with similar characteristics and time course. The current was highly Ca(2+)-selective and showed strong inward rectification, all typical of I(crac). The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of I(crac), and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited I(crac) activation in DT40(IP(3)R-/-) cells, even after complete depletion of intracellular Ca(2+) stores by ionomycin. Wild-type and DT40(IP(3)R-/-) cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40(IP(3)R-/-) cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP(3)Rs and RyRs can efficiently gate I(crac) in DT40 cells and explain the persistence of I(crac) gating by internal stores in the absence of IP(3)Rs.

Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells

Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca2+-release activated Ca2+ current (Icrac) by conformational coupling to Ca2+-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca2+ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course. The current was highly Ca2+-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.

Lack of direct role for calcium in ischemic diastolic dysfunction in isolated hearts.

Ischemia is characterized by an increase in intracellular calcium and occurrence of diastolic dysfunction. We investigated whether the myocyte calcium level is an important direct determinant of ischemic diastolic dysfunction. We exposed isolated, perfused isovolumic (balloon in left ventricle) rat and rabbit hearts to low-flow ischemia and increased extracellular calcium (from 1.5 to 16 mmol/L) for brief periods. Intracellular calcium was measured by aequorin. Low-flow ischemia resulted in a 270% increase (P:<0.05) in diastolic intracellular calcium, a 50% (P:<0.05) calcium transient amplitude decrease, and a 52% (P:<0.05) slowing of calcium transient decline. Diastolic pressure increased by 6+/-2 mm Hg (P:<0.05), and rate of systolic pressure decay decreased by 65% (P:<0.05). Experimentally increasing extracellular calcium doubled both intracellular diastolic calcium and calcium transient amplitude, concomitant with a developed pressure increase; however, there was no increase in ischemic diastolic pressure, slowing of the calcium transient decay, or further slowing of systolic pressure decay. Similarly, after 45 minutes of low-flow ischemia, after diastolic pressure had increased from 8.5+/-0.6 to 19.7+/-3.5 mm Hg (P:<0.001), intracoronary high-molar calcium chloride infusion increased systolic pressure from 36+/-4 to 63+/-11 mm Hg (P:<0.001), indicating an increase in intracellular calcium, but it decreased diastolic pressure from 19. 7+/-3.5 to 17.5+/-3.7 mm Hg (P:<0.01). Conversely, EGTA infusion decreased systolic pressure, indicating a decrease in intracellular calcium, but did not decrease diastolic pressure. When calcium availability was experimentally altered during ischemia, there was no alteration in left ventricular diastolic pressure, suggesting that ischemic diastolic dysfunction is not directly mediated by a calcium activated tension.

PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands

The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors, and a feedback regulatory mechanism of PTH on the secretion of PTH has been suggested. Because of the existence of a common receptor between PTH and PTH-related peptide (PTHrP), the aim of the present study was to examine the possible effects of PTHrP 1-40 and 1-86 on PTH secretion in rats. In vivo, the effect of PTHrP on Ca++-regulated PTH secretion was examined by the induction of hypocalcemia and hypercalcemia by an infusion of EGTA and Ca++, with and without PTHrP. The eventual effects of PTHrP on the peripheral metabolism of PTH were examined by infusion of human PTH (hPTH) with and without PTHrP. hPTH was measured by an intact hPTH assay not cross reacting with rat PTH or PTHrP. To examine whether near physiological levels of circulating PTH have an autoregulatory effect in vivo on PTH secretion from the parathyroid gland, an acute reduction of the circulating PTH was induced by an acute unilateral parathyroidectomy (UPTX). PTH secretion from the remaining parathyroid gland was followed in response to EGTA-induced hypocalcemia. In vitro investigations on the effect of PTHrP 1-40 on PTH secretion from whole rat parathyroid glands incubated in media containing a calcium concentration of 0.6 or 1.35 mmol/L were performed to confirm whether the effect of PTHrP was directly on the gland. The rat PTH assay was examined for cross reaction with PTHrP. In vivo, the same rate of decrease of plasma Ca++ was induced in the experimental groups. The maximal response of PTH to hypocalcemia (218 +/- 16 pg/mL, N = 6) was significantly enhanced by PTHrP 1-40 (525 +/- 79 pg/mL, N = 6) and by PTHrP 1-86 (465 +/- 29 pg/mL, N = 6, P < 0.001). No effect of PTHrP on PTH secretion was found during normocalcemia or hypercalcemia. UPTX resulted in a 50% reduction of PTH secretion, and no compensatory increase of PTH was observed. PTHrP had no effect on the metabolism of PTH. In vitro, low-Ca++-induced PTH secretion was significantly augmented by 300% (P < 0.01) when the medium contained PTHrP 1-40. PTHrP did not cross react with the PTH assay. PTHrP significantly enhanced the low-Ca++-stimulated PTH secretion in vivo and in vitro. An autocrine/paracrine role of PTHrP in the parathyroid glands is suggested. An autoregulatory effect of circulating PTH on the PTH secretion from parathyroid glands seems unlikely. The "maximal secretory capacity" of the parathyroid glands induced by hypocalcemia in vivo and in vitro is not the maximum, as PTH secretion can be increased even further, by several-fold.

1,25(OH) 2D 3 only affects long-term levels of plasma Ca 2+ but not the rapid minute-to-minute plasma Ca 2+ homeostasis in the rat

Results from our lab have shown previously that parathyroid hormone (PTH) is not the key factor in the rapid regulation of plasma Ca 2+. The possible role of 1,25(OH) 2D 3 in the rapid minute-to-minute regulation of plasma Ca 2+, as addressed by a possible rapid non-genomic action of 1,25(OH) 2D 3, was therefore studied in vivo in rats. The rapid calcemic recovery from induction of hypocalcemia by a brief EGTA infusion was examined in vitamin D-depleted rats with intact parathyroid glands and in vitamin D depleted rats 1 h after parathyroidectomy (PTX). The influence of different levels of plasma 1,25(OH) 2D 3 on the rapid calcemic recovery from hypocalcemia was examined in PTX rats treated with 1,25(OH) 2D 3 for two days at two different doses of 0.2 μg/day, 0.05 μg/day or vehicle, and in PTX rats being BNX for two days, as well. Additionally, the long-term effect of 1,25(OH) 2D 3 on plasma Ca 2+ homeostasis was examined. Plasma Ca 2+ recovered significantly ( P < 0.05) 10 min after discontinuing EGTA in vitamin D-depleted rats with or without parathyroid glands. Plasma Ca 2+ increased significantly ( P < 0.05) and at the same rate after induction of hypocalcemia in PTX rats with different levels of plasma 1,25(OH) 2D 3. The final levels of plasma Ca 2+ obtained were set by 1,25(OH) 2D 3 in a dose-related manner. 1,25(OH) 2D 3 did not affect the rapid calcemic recovery from EGTA induced hypocalcemia, but only had an effect on the long-term plasma Ca 2+ homeostasis in the rat.

Calciotrophic hormones during experimental hypocalcaemia and hypercalcaemia in spontaneously diabetic rats.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) concentrations have been found to be decreased in diabetic humans and rats. To investigate further the regulation of plasma Ca in diabetes, first we measured Ca(2+), P, Mg, parathyroid hormone(1-34) (PTH), and total and free 1,25(OH)(2)D(3) in male spontaneously diabetic rats 7 and 28 days after the onset of glycosuria. Secondly, we studied changes in the levels of PTH and 1,25(OH)(2)D(3) in response to hypocalcaemia induced by an i.v. infusion of EGTA (2.5%, wt/vol.) for 24 h, and changes in the levels of 1,25(OH)(2)D(3) in response to an i.v. infusion of rat PTH (10 microgram over 24 h) without or with concomitant EGTA infusion (producing hypercalcaemia or normo/hypocalcaemia respectively), in diabetic and control rats. Ca(2+), P, Mg and PTH concentrations remained within the control ranges after 7 and 28 days of glycosuria; 1,25(OH)(2)D(3) concentrations were decreased after 7, but not after 28, days of glycosuria. PTH concentrations showed a similar rise during EGTA-induced hypocalcaemia in control and diabetic rats compared with saline-infused rats, whereas 1,25(OH)(2)D(3) concentrations were unchanged in both groups. Total and free 1,25(OH)(2)D(3) levels were comparably (about 3-fold) increased during PTH, but not during combined PTH and EGTA infusion in control and diabetic rats. Total 1, 25(OH)(2)D(3) concentrations were lower in the diabetic groups infused with saline or PTH than in their respective controls, and there was a similar trend in the PTH+EGTA-infused group; free 1, 25(OH)(2)D(3) levels, however, were normal or increased in the diabetic groups, confirming our previous data. The novel finding of this study is that, despite severe insulin deficiency and altered 1, 25(OH)(2)D(3) levels, the in vivo response of PTH levels to hypocalcaemia and the in vivo response of 1,25(OH)(2)D(3) levels to PTH in diabetic rats are comparable with those found in nondiabetic rats.

Rapid recovery of plasma ionized calcium after acute induction of hypocalcaemia in parathyroidectomized and nephrectomized rats.

Plasma ionized calcium (Ca2+) is extremely tightly regulated in normal mammals. Even a small decline in Ca2+ is followed by a fast and steep increase of the parathyroid hormone (PTH) secretion and the current understanding of the calcium homeostasis indicates that PTH is the main factor responsible for this tight minute-to-minute regulation of the normal plasma Ca2+ concentration. However, experiments from our laboratory and some clinical experiences points towards the existence of factors, other than PTH, involved in the rapid minute-to-minute calcium homeostasis. Thus, the aim of the present study was to examine whether PTH plays an important role in the rapid upregulation of plasma Ca2+ after induction of hypocalcaemia in the rat. I. Parathyroidectomy (PTX) was performed in seven rats; 60 min later no PTH was detectable in the circulation. Then by a brief infusion of EGTA plasma Ca2+ was reduced from 1.26+/-0.02 to 0.86+/-0.02 mmol/l, P<0.001. Despite there being no PTH in the circulation plasma Ca2+ increased significantly to 0.97+/-0.02 mmol/l already 10 min after discontinuation of the EGTA infusion, P<0.04, and plasma Ca2+ was normalized within another 2 h. II. To evaluate a possible role of renal Ca2+ handling in the rapid upregulation of plasma Ca2+ a group of eight rats had acute PTX and bilateral nephrectomy (NX) performed; 60 min later plasma Ca2+ was reduced from 1.18+/-0.01 to 0.86+/-0.02 mmol/l by an EGTA infusion. Despite there being no PTH and no kidneys present plasma Ca2+ increased significantly already 10 min after discontinuation of EGTA to 0.96+/-0.02 mmol/l, P<0.02. After another 1.5 h the plasma Ca2+ reached the levels of the PTX/NX control rats. III. In order to exclude a possible action of receptor-bound PTH, which may have lasted for more than 1 h, seven rats were PTX 24 h before the induction of hypocalcaemia. Basal plasma Ca2+ was significantly reduced to 1.07+/-0.01 mmol/l, P<0.01. Then plasma Ca2+ was further reduced to 0.79+/-0.03 mmol/l by EGTA. Ten minutes after discontinuing EGTA plasma Ca2+ increased to 0.91+/-0.02 mmol/l, P<0.03 and 60 min later plasma Ca2+ reached the level of the control PTX rats. Normal rats with intact parathyroid glands had an exactly similar response of plasma Ca2+ to EGTA as that of 24 h PTX rats, but at significantly higher levels of plasma Ca2+ with a fall from 1.28+/-0.01 to 0.96+/-0.03 mmol/l and again a significant increase of plasma Ca2+ to 1.13+/-0.03 (P<0.001) 10 min after discontinuation of EGTA. After another hour basal levels were reached. Despite there being no PTH in the circulation a rapid increase of plasma Ca2+ occurs immediately after a brief induction of hypocalcaemia. The kidneys are not responsible for this phenomenon. The present results suggest the existence of a mechanism other than the effect of PTH, which is responsible for the rapid minute-to-minute regulation of plasma Ca2+ in the rat.

In vivo release of somatostatin from rat hippocampus and striatum

Rats were implanted with microdialysis probes in hippocampi and striata, and somatostatin-like immunoreactivity (SS-LI) was measured in outflows obtained from awake, freely moving animals 48 and 72 h post implantation. SS-LI was measurable in all dialysates under basal conditions; concentrations were stable and within a narrow range, about 3–6 fmol/ml. Cysteamine (300 mg/kg, s.c.) markedly reduced basal SS-LI concentrations in outflows from hippocampus ( P < 0.00001). KCl (100 mM, 10 min) or veratridine (50 μM, 10 min) infusion elevated hippocampal SS-LI output by 55 and 106%, respectively ( P's < 0.05). EGTA (10 mM) or tetrodotoxin (2 μM) infusion inhibited the SS-LI release elicited by KCl and veratridine, respectively, without affecting the basal SS-LI outflow. Thus, our results demonstrate that SS-LI is released from rat hippocampus and striatum in vivo, and provide evidence that the peptide may be released in hippocampus by both action potential dependent and independent processes.

Role of cyclic nucleotides and calcium in the nutrient-induced release of cholecystokinin-like immunoreactivity in rats.

1. This study was undertaken with an isolated vascularly perfused rat duodenojejunal preparation to investigate the mechanisms of the release of cholecystokinin measured by immunoassay (cholecystokinin-like immunoreactivity, CCK-LI). 2. Intra-arterial infusion of forskolin (2-20 microM) evoked a prompt and well-sustained secretion of CCK-LI which was increased to a mean value of 600% of basal with the highest dose tested. 3-Isobutyl-1-methylxanthine (IBMX) (10(-6)-10(-4) M) stimulated the secretion of CCK-LI (maximal increase of 400% of basal at 10(-4) M). 3. Intra-arterial infusion of beta-phorbol 12-myristate 13-acetate (5 x 10(-7)-5 x 10(-6) M) and calcium ionophore A23187 (10(-6)-10(-5) M) alone or in combination provoked only a transient increase in the release of CCK-LI. 4. Luminal infusion of a 5% ovalbumin hydrolysate solution produced an immediate release of CCK-LI followed by a well-sustained secretion at 580% of basal. Intra-arterial infusion of IBMX (10(-5) or 10(-4) M) in combination with luminal peptone induced a release of CCK-LI which was equal to the sum of the CCK responses evoked by IBMX and peptone given separately. 5. Intra-arterial infusion of EGTA (2 mM) abolished the forskolin- and peptone-induced CCK secretion while luminal EGTA (2 mM) had no inhibitory effect. Verapamil (5 x 10(-5)-10(-4) M) or nifedipine (10(-5)-5 x 10(-5) M) inhibited the peptone-evoked CCK secretion. A high concentration of trifluoperazine (10(-4) M) strongly reduced the release of CCK-LI induced by intraluminal peptone while 10(-5) M was ineffective. 6. It is concluded that the peptone-induced secretion of CCK-LI involves a cyclic AMP-dependent mechanism and the activation of calcium channels possibly located at the basolateral side of the CCK cell.

Mechanism of the effect of exogenous fructose 1,6-bisphosphate on myocardial energy metabolism.

The effects of fructose 1,6-bisphosphate (F-1,6-P2) on the isolated Langendorff-perfused heart were studied by monitoring flavoprotein fluorescence, oxygen consumption (MVO2), coronary flow (Fc), systolic intraventricular pressure (Psys), diastolic intraventricular pressure, and contraction frequency. The cellular energy state and cytosolic pH were determined by means of 31P nuclear magnetic resonance. Infusion of 5 mM F-1,6-P2 caused a rapid shift toward reduction in the flavoprotein redox state and initial 50% and 44% decreases in Psys and MVO2, respectively. After a partial recovery, these measures remained 11% and 25% below the basal value. Concomitantly, after an initial transient increase of 13%, Fc remained 17% lower than in the basal state. When the F-1,6-P2 concentration was subsequently increased to 10 mM, psys and MVO2 dropped temporarily to 31% and 29% of the basal value and then remained at 50% and 53%, respectively. Simultaneously, a brief increase was observed in Fc, which then fell 34% below the basal value. Rapid reoxidation of the flavoproteins and increases in MVO2, Psys, and Fc occurred on discontinuation of the F-1,6-P2 infusion. 31P nuclear magnetic resonance during infusions of both 5 and 10 mM F-1,6-P2 revealed a decrease in cytosolic inorganic phosphate and a tendency to increase creatine phosphate, suggesting elevation in the cellular energy state. No changes in intracellular pH occurred as estimated from the chemical shift of the nuclear magnetic resonance of inorganic phosphate. F-1,6-P2 (5 mM and 10 mM) lowered the free Ca2+ concentration in the Krebs-Henseleit bicarbonate buffer (by 32% and 47%, respectively). This probably explains the effects of F-1,6-P2 on mechanical work performance and cellular respiration. A direct metabolic effect also exists, however, because flavoprotein reduction by F-1,6-P2 could be observed in the K(+)-arrested heart, where its effects on MVO2 were minimal. This redox effect may not be caused by changes in free Ca2+ concentration because it could not be reproduced by infusion of EGTA.

Regulation of parathyroid hormone secretion by plasma calcium in aging rats

Plasma immunoreactive parathyroid hormone (irPTH) levels increase with aging. This study determined 1) whether NH2-terminal irPTH secretory responses to induced hypocalcemia differ between adult (6-mo-old) and aged (24- to 26-mo-old) male rats and 2) whether a higher set point for irPTH release by Ca is responsible for the elevated irPTH levels with aging. Basal irPTH levels were 68% higher and 1,25-dihydroxyvitamin D3 levels were 44% lower in aged rats. An acutely induced, constant hypocalcemic stimulus [0.32 mM decrement in ionized Ca (Ca2+) for 2 h] was developed in catheterized conscious adult and aged rats by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) infusion using the Ca clamp technique. The initial irPTH secretory response to acute hypocalcemia (5-10 min) was reduced in aged rats (1.9- vs. 3.1-fold increase), suggesting reduced hormone stores. However, higher sustained irPTH levels (30 min to 2 h) were maintained in aged rats, indicating increased irPTH synthesis and release. The EGTA infusion rate necessary to maintain constant hypocalcemia was less in aged rats, suggesting skeletal resistance to PTH. Slow EGTA and Ca infusions were used to determine irPTH secretion at plasma Ca2+ levels from 0.7 to 1.5 mM. In aged rats, irPTH levels were higher at all Ca2+ concentrations, but the set point for irPTH release by Ca2+ was the same as in adult rats. Thus the elevated irPTH secretion in aged rats is not caused by a change in the set point for irPTH release but does result in decreased irPTH stores.

Calcium administration increases the mortality of endotoxic shock in rats

Calcium chloride is administered frequently to critically ill patients to improve cardiac output and BP. However, Ca has been implicated in the pathophysiology of shock and ischemic disorders. To test the hypothesis that Ca may be deleterious to shock outcome, we studied the effects of CaCl and Ca chelator (EGTA) infusions on mean arterial pressure (MAP) responses to endotoxin and 24-h survival in rats. Increasing ionized Ca from 4.1 +/- 0.06 to 4.9 +/- 0.20 and 8.5 +/- 0.52 mg/dl progressively increased endotoxin lethality from 20% to 37% and 80%, respectively. This occurred despite slight improvements in MAP in hypercalcemic rats. Conversely, hypocalcemia (3.6 +/- 0.08 mg/dl) lowered endotoxin-induced mortality to 0 without significant effects on MAP. Ca and EGTA infusions alone were not associated with any mortality. Although Ca administration may improve MAP, it significantly increases mortality associated with endotoxic shock in rats. Based on these observations, we advise caution when using Ca in patients with sepsis.

Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period.