American Journal of Physiology-Legacy Content

Published by American Physiological Society

Print ISSN: 0002-9513

Articles


Levels and reduction
  • Article

October 1977

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62 Reads

This paper addresses the problem of reconciling pluralism with reductionism, i.e., acknowledging both the variety of the world and the need and possibility to explain it. First the various kinds of monism and pluralism that litter the scientific and philosophical literature are examined cursorily. Then certain maligned notions are examined, mainly those of novelty, self-assembly, level, and levels "hierarchy." They are shown to be amenable to analysis and even mathematization. Then the logic of reduction is analyzed. Two kinds of reduction are distinguished: full or straight, and partial or roundabout. And three stands on reduction are examined: anti-, radical, and moderate reductionism. The former is dismissed for being obscurantist and the second for being quixotic. Moderate reductionism, aiming at the (partial) reduction of higher levels to lower ones without skipping any intermediate levels, is adopted. Finally moderate reductionism is found to be consistent with a certain variety of pluralism, characterized as naturalistic.
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Heart rate response to hemorrhage-induced 0.05-Hz oscillations in arterial pressure in conscious dogs

May 1991

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44 Reads

We have previously reported that oscillations at 0.05 Hz can be generated by a simple computer model incorporating a negative-feedback reflex mechanism and an effector mechanism with a time delay. Computer simulations by inhibiting the vagal effector mechanism and activating the adrenergic effector mechanism elicited low-frequency oscillations at a frequency of 0.05 Hz in heart rate. We have observed that the cardiovascular system of the conscious dog, when stressed by the loss of blood, generates oscillations in arterial pressure and heart rate at a frequency of 0.05 Hz. We investigated in six conscious dogs the role of the sympathetic and parasympathetic nervous systems in generating these heart rate oscillations. During baseline conditions, the predominant peak in the arterial pressure and heart rate power spectra was located at the respiratory frequency, while the low-frequency oscillations were small. After a 30-ml/kg hemorrhage or after an 8-, 15-, or 30-ml/kg hemorrhage with glycopyrrolate, a muscarinic-blocking agent, low-frequency oscillations at a frequency of 0.05 Hz predominated, while the respiratory frequency oscillations were negligible. Since respiratory frequency oscillations have been reported to reflect vagal activity, and since the low-frequency oscillations were present after vagal blockade, these hemorrhage-induced low-frequency oscillations in heart rate may be primarily mediated by the cardiac sympathetic nerves. Also cross-correlation analysis between arterial pressure and heart rate showed that a change in arterial pressure caused an opposite change in heart rate with a delay of 2-5 s. We conclude that hemorrhage-induced oscillations in heart rate at 0.05 Hz represent the arterial baroreceptor-beta-sympathetic reflex response to underlying arterial pressure oscillations.

Endogenous nitric oxide buffers blood pressure variability between 0.2 and 0.6 Hz in the conscious rat

February 1997

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36 Reads

Shear stress is a potent stimulus for the formation and release of nitric oxide (NO). It seems, therefore, possible that a short-term increase in arterial blood pressure (ABP), which leads to a concomitant rise in endothelial shear stress, enhances NO release. The latter elicits a relaxation of vascular smooth muscle cells that, in turn, counteracts the initial rise in blood pressure (BP). Thus this chain of events may constitute a negative feedback loop reducing BP variability (BPV). To test this hypothesis, BP-time series were determined via telemetry in freely moving conscious Sprague-Dawley rats. Because it was reported recently that NO effects on ABP are more pronounced in females, the experiments were performed on 2 groups consisting of 10 female and 11 male animals. This was done under control conditions and after fixing NO plasma levels via an intravenous bolus of 15 mg/kg body wt N(G)-nitro-L-arginine methyl ester together with a continuous infusion of nitroprusside (15 ± 0.8 μg/min). This combined infusion maintained mean ABP and heart rate at physiological levels, thus avoiding as much as possible interferences with other reflexes, e.g., the baroreflex. To quantitate BPV, fast Fourier transforms of the BP-time series were determined. The absolute power in the frequency range below 1 Hz increased during fixed NO to ~350% vs. control animals (female control, 2.1 x 109 ± 1.5 x 108 mmHg2 vs. fixed NO, 8.0 x 109 ± 1.3 x 109 mmHg2, P < 0.005; male control, 3.4 x 109 ± 4.6 x 108 mmHg2 vs. fixed NO, 8.3 x 109 ± 2.0 x 109 mmHg2, P < 0.05). This was mainly caused by a substantial rise in the power ranging from 0.2 to 0.6 Hz, which increased roughly fourfold in both females and males. It is concluded that the NO system is a potent buffer of spontaneous BP oscillations in the freely moving rat. This system is most efficient in buffering frequencies within the range of 0.2- 0.6 Hz and shows no gender-specific differences with respect to its BP buffering capacity.

Kinetics of glucose delivery to subcutaneous tissue in rats measured with 0.3-mm amperometric microsensors

August 1995

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The time between intravenous injection of a glucose bolus and the time the glucose concentration peaked in the subcutaneous tissue was measured in pentobarbital-anesthetized rats with implanted 290-microns-diameter amperometric sensors. Boluses of 100, 200, and 400 mg/kg body wt were injected. The glucose concentration in the jugular vein was monitored by frequent withdrawal and analysis of samples. The glucose concentration in the subcutaneous tissue was continuously monitored with the sensors. The times required for the subcutaneously implanted sensor to reach its maximum current, corrected for sensor response times, were 7.5 +/- 3.9, 9.8 +/- 5.5, and 10.0 +/- 4.4 min for the smallest to the largest dose, respectively. The shorter delay in response to the smallest dose was statistically significant (P < 0.03). The results were consistent with dilution of the bolus in the cardiovascular system and transport of glucose by both diffusion and facilitated transport via a saturable mediator. An understanding of the differences in the dynamics of venous vs. subcutaneous response to a glucose dose is important in developing algorithms for the control of blood glucose based on a subcutaneous measurement.

Effect of maternal glucocorticoid treatment on fetal fluids in sheep at 0.4 gestation

May 1994

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9 Reads

Treatment of nine pregnant Merino ewes (64.0 +/- 0.4 days of gestation) with dexamethasone (D; 0.76 mg/h for 48 h) resulted in significant alterations in fetal fluids compared with eight saline-infused control animals (S; 63.0 +/- 0.9 days). There was a substantial increase in allantoic fluid volume (177 +/- 18 ml, D vs. 31 +/- 6, S) but no change in amniotic fluid volume (248 +/- 12 ml, D; 305 +/- 24, S). For allantoic fluid there was a significant decrease in osmolality (213 +/- 4 mosmol/kg water, D; 230 +/- 5, S) and alterations in composition. Amniotic fluid osmolality was unchanged (292 +/- 2 mosmol/kg water, D; 293 +/- 1, S), but amniotic fluid composition was affected. In four fetuses in which bladder and amniotic cannulas were inserted at gestational age 68-75 days, fetal urine flow rate increased from a mean of 4.1 +/- 1.1 to 13.8 +/- 2.6 ml/h after 24 h and 11.8 +/- 3.0 ml/h at 48 h for a similar maternal D infusion, whereas no such increase occurred in four control fetuses. All the fetal urine voided during a 3.5- to 4-h infusion of 51Cr-labeled EDTA into the fetal bladder was directed to the allantois. The results suggest that the increase in allantoic fluid volume resulted from increased fetal urine output into the allantoic compartment, although the composition of the excess allantoic fluid differed substantially from that of fetal urine. There was a greater incidence of abnormal cotyledons in the D-infused ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Sympathetic activity and blood pressure are tightly coupled at 0.4 Hz in conscious rats

December 1994

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12 Reads

Interactions of sympathetic nerve activity (SNA) with blood pressure (BP) and heart rate (HR) were assessed in conscious rats while they rested quietly in a cloth sock (n = 7), roamed freely in their home cage (n = 6), and then after anesthesia with pentobarbital (30 mg/kg; n = 7). The power and coherence spectra below 3 Hz were calculated from data collected for 9.56 min. In the conscious rat, SNA spectral power peaked at 0.4 Hz, whereas the majority of spectral power for both BP and HR occurred at frequencies lower than 0.4 Hz. However, there was an inconspicuous peak in the BP power spectra at 0.4 Hz that was not seen in the HR spectra. Coherence between SNA and BP peaked at a frequency of approximately 0.4 Hz, the same frequency at which the SNA spectral peaks occurred. In contrast, at frequencies below 0.4 Hz where maximum BP power occurred, the coherence was considerably lower. Anesthesia with pentobarbital lowered spectral power for BP, SNA, and HR but essentially did not change the coherence between SNA and BP. Interactions between respiration and each of the other variables were weak in the conscious rat. However, prominent respiratory interactions at approximately 1.2 Hz were evident after anesthesia. These data indicate a close coupling between SNA and BP at 0.4 Hz, raising the possibility that the BP spectral power at 0.4 Hz reliably reflects sympathetic activity.

First-order differential-delay equation for the baroreflex predicts the 0.4-Hz blood pressure rhythm in rats

January 1998

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29 Reads

We have described a 0.4-Hz rhythm in renal sympathetic nerve activity (SNA) that is tightly coupled to 0.4-Hz oscillations in blood pressure in the unanesthetized rat. In previous work, the relationship between SNA and fluctuations in mean arterial blood pressure (MAP) was described by a set of two first-order differential equations. We have now modified our earlier model to test the feasibility that the 0.4-Hz rhythm can be explained by the baroreflex without requiring a neural oscillator. In this baroreflex model, a linear feedback term replaces the sympathetic drive to the cardiovascular system. The time delay in the feedback loop is set equal to the time delay on the efferent side, approximately 0.5 s (as determined in the initial model), plus a time delay of 0.2 s on the afferent side for a total time delay of approximately 0.7 s. A stability analysis of this new model yields feedback resonant frequencies close to 0.4 Hz. Because of the time delay in the feedback loop, the proportional gain may not exceed a value on the order of 10 to maintain stability. The addition of a derivative feedback term increases the system's stability for a positive range of derivative gains. We conclude that the known physiological time delay for the sympathetic portion of the baroreflex can account for the observed 0.4-Hz rhythm in rat MAP and that the sensitivity of the baroreceptors to the rate of change in blood pressure, as well as average blood pressure, would enhance the natural stability of the baroreflex.

Rat lung metabolism after 3 days of continuous exposure to 0.6 ppm ozone

March 1986

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10 Reads

Continuous exposure of rats to low concentrations of ozone has previously been associated with enhanced metabolic enzyme activities, when measured in lung homogenates. In this study, metabolic rates were measured in intact perfused lungs with altered pathology brought about by 3 days continuous exposure to 0.6 ppm ozone. Increased metabolism of ozone-exposed lungs was indicated by a twofold enhancement in glucose utilization, associated with a 62% increase in lactate formation and a 166% increase in the rate of 14CO2 production from D-[U-14C]glucose from control values of 5.2 +/- 0.5 mumol lactate and 4.4 +/- 0.6 mumol 14CO2/h per lung (+/- SE, n = 4), respectively. Mitochondrial metabolism was separately assessed by measurements of 14CO2 production from [U-14C]-pyruvate, which was found not to be significantly altered by ozone exposure, although homogenate oxygen uptake in the presence of succinate was significantly enhanced by 57%. These changes in intermediary metabolism could be correlated with increased glucose carbon incorporation into lipid and elevated activity of glucose-6-phosphate dehydrogenase. The observed elevated metabolic rates were consistent with the energy and synthetic needs of a lung during repair of ozone-induced damage.

Incorporation of glucose carbons into rat lung lipids after exposure to 0.6 ppm ozone

June 1985

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8 Reads

Continuous exposure to low concentrations of ozone has previously been associated with proliferation of lung alveolar type II epithelial cells. In this study, 14C incorporation into tissue lipids was determined in isolated rat lungs by perfusion with [U-14C]glucose, at a time of maximal hyperplasia brought about by 3 days continuous exposure to 0.6 ppm ozone. Ozone exposed lungs exhibited increased rates of glycolytic energy production, indicated by an 89% increase in 3H2O generation on perfusion with [5-3H]glucose, from a control value of 17.5 +/- 2.1 mumol X h-1 X g-1 X dry wt-1 (+/- SE, n = 4). Ozone exposure resulted in enhanced 14C incorporations into glyceride-glycerol and fatty acid moieties of lung lipids of 95 and 180%, respectively, with a greater proportion of label being recovered in shorter chain fatty acids. Although increased labeling was observed in both neutral and phospholipids, the pattern of 14C recovery suggested a relative increased glucose carbon incorporation into lung free fatty acids, phosphatidic acid, and such membrane associated lipids as phosphatidylinositol and those containing sphingosine. These results are consistent with the needs of a dividing cell population for enhanced energy production and synthesis of new lipids.

Cerebrovascular reactivity to adenosine analogues in 0.6-0.7 gestation and near-term fetal sheep

June 1992

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During acute hypoxia, fetal sheep less than 0.7 gestation increase cerebral blood flow (CBF) relatively less than fetal sheep near term. We hypothesized that cerebrovascular reactivity to a hypoxic vasodilator metabolite such as adenosine might be diminished in immature fetuses. This study examined cerebral vasoreactivity to adenosine analogues in nine sheep fetuses less than 0.7 gestation (90-103 days) and nine near term (129-143 days). Fetuses were equipped in utero with a closed cranial window, and pial arterioles were studied by intravital microscopy. 5'-(N-ethylcarboxamido)-adenosine (NECA; 10(-9)-10(-5) M) and N6-cyclopentyladenosine (CPA; 10(-9)-10(-4) M) each caused a dose-dependent increase in arteriolar diameter that was attenuated in the presence of the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 5 x 10(-6) M). Dose-response curves to the agonists were similar for both age groups. NECA was a more potent vasodilator than CPA, in keeping with their affinity for the A2 receptor. Suffusion of 8-PT alone at less than 10(-5) M had no effect on arteriolar diameter. We conclude that adenosine is able to dilate fetal cerebral arterioles as young as 0.6 gestation by acting at an A2 receptor, although resting tone is not influenced by adenosine. The immature fetal sheep CBF response to hypoxia is not attributable to undeveloped vasoreactivity to adenosine.

Responses to acute hypoxemia in fetal sheep at 0.6-0.7 gestation

April 1989

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12 Reads

A majority of previous studies of fetal responses to acute hypoxemia has focused on the response of the sheep fetus greater than 120 days of gestation when many regulatory systems have been established. To assess the response of younger, less well-developed fetuses, we exposed two groups of fetal sheep (I, 84-91 days; II, 97-99 days gestational age) to acute hypoxemia by giving the ewe a gas mixture containing 9% O2 to breathe. We decreased descending aortic PO2 in both groups of fetuses [I, 24 +/- 6 to 14 +/- 3 (SD) Torr; II, 23 +/- 3 to 12 +/- 4 Torr] by a degree similar to that achieved in previous studies of fetuses greater than 120 days of gestation. Mean arterial blood pressure (I, 31 +/- 6; II, 40 +/- 3 Torr) did not change significantly from control values, and heart rate (I, 224 +/- 27; II, 203 +/- 16 beats/min) increased significantly in group II fetuses with hypoxemia. In group I and II fetuses, as in older fetuses, cerebral, myocardial, and adrenal blood flows, measured by the microsphere technique, increased, and pulmonary blood flow decreased. These responses mature early and are likely local vascular responses to decreases in oxygen content. Combined ventricular output and umbilical-placental blood flow decreased significantly in both groups. Unlike the response of the fetus greater than 120 days, acute hypoxemia did not decrease blood flow to the musculoskeletal and cutaneous circulations (group I only), gastrointestinal, or renal circulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of graded umbilical cord compression in fetal sheep at 0.6-0.7 gestation

November 1991

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4 Reads

To define responses of immature fetuses to asphyxia, we occluded the umbilical cord of 11 chronically instrumented fetal sheep at 82-94 days gestation and measured hemodynamic and catecholamine responses. The fetuses became acidemic, hypoxemic, and hypercarbic: arterial pH and PO2 decreased from 7.36 +/- 0.04 and 22 +/- 3 Torr to 7.10 +/- 0.04 (mean +/- SD, P less than 0.01) and 15 +/- 4 Torr (P less than 0.01), respectively, and PCO2 increased from 56 +/- 5 to 86 +/- 8 Torr (P less than 0.01) when umbilical blood flow was reduced by 75-88%. This degree of reduction in umbilical blood flow decreased cardiac output from 606 +/- 101 to 247 +/- 67 ml.min-1.kg-1 (P less than 0.01) and blood flow to hepatic, renal, musculoskeletal, and pulmonary vascular beds. Plasma norepinephrine concentrations increased from 1,557 +/- 975 to 16,718 +/- 14,672 pg/ml (P less than 0.05) with a 75-88% reduction, but mean arterial blood pressure did not increase. The absence of a hypertensive response probably relates to the decrease in cardiac output. These data indicate that asphyxia severely compromises cardiac output and organ perfusion in the midgestation fetus.


Regulation of cell volume by the osteosarcoma cell line UMR-106-01

October 1991

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5 Reads

Determination of cell volume by an electronic cell-sizing technique was used to study the role of ion transporters in cell volume regulation by the osteosarcoma cell line UMR-106-01. Swelling the cells in hypotonic medium was followed by regulatory volume decrease (RVD). The rate of RVD was strongly dependent on the subpassage used and increased with increasing subpassages. Swelling-evoked changes in cytosolic free Ca2+ ([Ca2+]i) did not account for this behavior, since it was similar in cells from all subpassages. Increasing plasma membrane K+ permeability with valinomycin resulted in a similar rate of RVD in cells from different subpassages, suggesting increased K+ channel activity or other electrogenic transporter with increased subpassages. In contrast, the mechanisms responsible for regulatory volume increase (RVI) were fully active in cells from all subpassages. Increasing medium osmolarity of cells bathed in isotonic medium induced slow and incomplete RVI. In addition, shrinking cells exposed to hypotonic medium before completion of RVD resulted in impaired RVI. Effective RVI could be observed only after completion of RVD of cells exposed to hypotonic medium. Removal of extracellular Na+ or K+ completely blocked RVI, whereas removal of external Cl- partially blocked RVI. The effect of K+ removal probably reflects in part inhibition of Na-K-2Cl cotransport and in part inhibition of the Na+ pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone increases Gα(q-11) expression and hormone stimulated phospholipase c activity in UMR-106-01 cells

September 1997

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10 Reads

Glucocorticoids regulate responsiveness of many cells to hormones that bind to G protein-coupled receptors. We examined the effect of glucocorticoids on parathyroid hormone (PTH) activation of two G protein-activated signal transduction pathways, phospholipase C (PLC) and adenylyl cyclase, in osteosarcoma UMR-106-01 cells. Dexamethasone (100 nM) increased PTH-stimulated and NaF-stimulated PLC activity by > 100% over 4 days (223 +/- 8 and 293 +/- 8.2% of control after 4 days for PTH and NaF-stimulated activity, respectively). The increase in PTH-stimulated adenylyl cyclase response in the same cells was more modest (162 +/- 5.4 and 171 +/- 6.8% of control after 4 days for PTH and NaF-stimulated activity, respectively). PTH activation of PLC was blocked by antiserums to G alpha q-11 and activation of adenylyl cyclase by G alpha s antiserums. Quantification of these G protein subunits in control and dexamethasone-treated cells showed a 78% increase in G alpha q-11 (from 18.1 +/- 1.2 to 32.2 +/- 1.5 pmol/mg), whereas G alpha s was increased only 34% (from 6.2 +/- 0.5 to 8.2 +/- 0.3 pmol/mg) and G beta-subunits were increased 40% (from 54 +/- 2.3 to 75.2 +/- 3.8 pmol/mg). These results suggest that glucocorticoids are more potent regulators of PLC activity than adenylyl cyclase activity in UMR cells, and this is mediated, at least in part, by differential increases in G alpha q-11 proteins.

Insulin-like growth factor I stimulates sodium-phosphate cotransport in the rat osteoblastic cell line UMR-106-01

May 1992

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The effect of recombinant insulin-like growth factor I (IGF-I) on Pi uptake by a rat osteoblast-like cell line (UMR-106-01) in culture was investigated. IGF-I (10(-6)-10(-8) M) caused a dose-related stimulation of Na(+)-Pi cotransport. A 30-70% increase in Na(+)-dependent Pi uptake over control values was observed after 1- to 5-h exposure of these cells to 10(-7) M IGF-I. The increase was detected within 45 min, in contrast to the slower action of insulin. This effect of IGF-I was specific for Na(+)-Pi uptake, because Na(+)-independent Pi uptake and Na(+)-alanine cotransport were unaffected by IGF-I. A reversal of IGF-I induced stimulation of Na(+)-Pi cotransport was observed within 1 h of removal of the hormone. Kinetic analysis of the IGF-I effect indicates a significant change only in the apparent maximum velocity (Vmax) of Na(+)-Pi cotransport. The Vmax was 5.22 +/- 0.47 vs. 3.33 +/- 0.45 nmol Pi.mg protein-1.10 min-1 in confluent monolayers exposed to 10(-7) M IGF-I and vehicle alone, respectively, for 3 h (P less than 0.05, group t test). Blocking de novo protein synthesis with cycloheximide had no effect on this stimulatory effect of IGF-I. These observations indicate that IGF-I specifically stimulates Pi uptake in osteoblastic cells. The effect is characterized by an increase in Vmax and is not dependent on de novo protein synthesis. The mechanism remains to be determined.

Beneficial actions of CP-0127, a novel bradykinin receptor antagonist, in murine traumatic shock

March 1994

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We studied the effects of CP-0127, a novel bradykinin receptor antagonist, in a rat model of traumatic shock. Pentobarbital-anesthetized rats subjected to Noble-Collip drum trauma developed a shock state characterized by marked hypotension, significant increases in plasma-free amino-nitrogen (8.6 +/- 0.97 vs. 2.3 +/- 0.15 U/ml in control rats) and intestinal myeloperoxidase (MPO) activity (2.7 +/- 0.33 vs. 0.08 +/- 0.03 U/100 mg control rats, intestinal tissue), and a survival time of only 110 +/- 9 min. Moreover, superior mesenteric artery (SMA) rings isolated from rats subjected to traumatic shock relaxed to the endothelium-dependent vasodilator acetylcholine (ACh) significantly less than rings isolated from control rats (21 +/- 4 vs. 92 +/- 4%, P < 0.001). Administration of CP-0127 at a dose of 10 mg/kg subcutaneously completely blocked the hypotensive response to 2.5 micrograms/kg bradykinin injected intravenously in sham traumatic shock rats. CP-0127 given immediately posttrauma prolonged survival time to 219 +/- 27 min (P < 0.01) and attenuated the increases in plasma-free amino-nitrogen (3.7 +/- 0.41 U/ml, P < 0.01) and tissue MPO activities (1.2 +/- 0.71 U/100 mg intestinal tissue, P < 0.05). Furthermore, SMA endothelial function was significantly preserved (relaxation to ACh: 57 +/- 6%, P < 0.01) in CP-0127-treated traumatic shock rats. These results indicate that bradykinin plays an important role in tissue injury associated with traumatic shock and that CP-0127 affords significant protection, which may be achieved through inhibition of neutrophil-endothelial interaction and protection of vascular endothelial function.


Novel recombinant serpin, LEX-032, attenuates myocardial reperfusion injury in cats

March 1996

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We studied the potential cardioprotective effects of the novel recombinant serine protease inhibitor (serpin), LEX-032, which inhibits the serine proteases elastase and cathepsin G. LEX-032 is a recombinant construct of human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of human alpha 1-protease inhibitor. Cats were subjected to 90 min of left anterior descending coronary artery (LAD) occlusion and 270 min of reperfusion (MI/R). Either LEX-032 or its vehicle (i.e., phosphate-buffered saline) was administered intravenously 10 min before reperfusion. Control cats were subjected to sham MI/R. Cats treated with LEX-032 demonstrated a marked reduction in cardiac necrosis after MI/R compared with cats receiving only vehicle (10 +/- 3 vs. 31 +/- 3%, P < 0.01). In addition, relaxation of LAD rings to the endothelium-dependent dilators (e.g., acetylcholine and A23187) was greater in the LEX-032-treated group than in cats receiving vehicle (72 +/- 5 vs. 52 +/- 7%, P < 0.05, and 74 +/- 8 vs. 50 +/- 8%, P < 0.05, respectively), indicating that endothelial function was preserved by LEX-032. Moreover, LEX-032 administration resulted in a marked reduction of polymorphonuclear leukocyte (PMN) adherence to ex vivo coronary vascular endothelium compared with vehicle (33 +/- 4 vs. 86 +/- 7 PMNs/mm2, P < 0.01). These data indicate that LEX-032 is a significant cardioprotective agent exerting its protective effect by inhibition of PMN-mediated cellular injury, and this agent represents a novel means of attenuating PMN-mediated reperfusion injury.

Dexamethasone rapidly increases hypothalamic neuropeptide Y secretion in adrenalectomized 06/06 mice

August 1996

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A single intracerebroventricular injection of dexamethasone (DEX) rapidly (within 30 min) suppresses brown adipose tissue thermogenesis and increases plasma insulin concentrations in adrenal-ectomized (ADX) ob/ob mice but not in ADX lean mice. Intracerebroventricular neuropeptide Y (NPY) administered intracerebroventricularly causes these same metabolic changes within 30 min in both ob/ob and lean ADX mice. We therefore hypothesized that DEX exerts these rapid-onset metabolic actions in ob/ob mice via a phenotype-specific enhancement of NPY secretion within the central nervous system. In support of this hypothesis, DEX (a type II glucocorticoid receptor agonist) administered intracerebroventricularly selectively lowered NPY concentrations in the whole hypothalamus of ADX ob/ob mice by 35% and in the arcuate nucleus region by approximately 70% within 30 min but not in the brain stem or hippocampus or in any of these regions of lean mice. DEX also functioned in vitro to enhance depolarization-dependent release of NPY from hypothalamic blocks of ADX ob/ob mice but not of ADX lean mice. Thus DEX acts in the hypothalamus of ob/ob mice in a phenotype-specific manner to evoke rapid transport of NPY from cell bodies within the arcuate nucleus to terminal regions including the dorsomedial and ventromedial hypothalamic regions for release.


1,2-Diacylglycerol and ceramide levels in rat liver and skeletal muscle in vivo

December 1991

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1,2-Diacylglycerol and ceramide levels were measured in liver and skeletal muscle of rats under the following four experimental conditions: 1) during rapid fetal growth in the second half of gestation and during postnatal aging, 2) during tissue anoxia lasting up to 10 min, 3) during fasting for up to 6 days, and 4) during stress induced by injection of endotoxin (lipopolysaccharide from Salmonella enteritidis). 1,2-Diacylglycerol and ceramide levels in fetal liver were 77 and 58% lower, respectively, than those of young rats after weaning. 1,2-Diacylglycerol and ceramide concentrations in liver of postnatal rats were not influenced by aging. Anoxia produced a 41-64% increase in liver 1,2-diacylglycerol levels and a 9-21% increase in liver ceramides. Fasting of adult rats for up to 6 days had no effect on 1,2-diacylglycerol and ceramide levels in liver. However, fasting increased skeletal muscle concentrations of 1,2-diacylglycerol and ceramides 88 and 44%, respectively. Injection of endotoxin had a biphasic effect on liver 1,2-diacylglycerol levels, causing a transient 52% increase at 1 h, followed by a 24-38% decrease below the control level at 17 h after endotoxin administration. Liver ceramide levels were increased 66, 52, and 27% at 1, 3, and 5 h after endotoxin, respectively, but did not differ from control at 17 h after injection. Endotoxin had no effect on muscle 1,2-diacylglycerol and ceramide concentrations at any interval.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, amiloride analogues, and renal Na+/H+ antiporter

April 1987

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N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a carboxyl-activating agent and has been shown to inhibit the renal Na+/H+ antiporter. The purposes of the present studies were to characterize the kinetics of inhibition of the Na+/H+ antiporter by EEDQ and to determine whether amiloride analogues affect the ability of EEDQ to inhibit the rate of Na+/H+ exchange. Brush-border membrane vesicles (BBMV) were prepared from rabbit kidneys; Na+/H+ exchange rate was assessed by the fluorescence quenching of acridine orange. EEDQ produced a concentration-dependent inhibition of Na+/H+ exchange; the effect was to decrease the maximum activity (Vmax) from 5.51 to 2.03 fluorescence units X mg protein-1 X S-1) and Km (from 14.1 to 8.7 mM) compared with control BBMV. Pretreatment of BBMV with amiloride before the addition of EEDQ maintained both Vmax and Km at values that were not significantly different from those for control BBMV. Compared with a series of analogues, amiloride was only the third most potent inhibitor of the rabbit renal Na+/H+ antiporter; amiloride, however, provided the greatest protection against inhibition of the antiporter by the subsequent addition of EEDQ. These findings suggest that the 2-carbonylguanidininum moiety and 6-chloro atom are important for binding of amiloride to sites at or near the antiporter; the group at position 5 is important in determining the ability of amiloride to protect against inhibition of the Na+/H+ antiporter by EEDQ. Finally, the ability of amiloride to protect against inactivation of the renal Na+/H+ antiporter by EEDQ is reversible.

1,2-dioctanoyl-sn-glycerol depresses cardiac L-type Ca2+ current: Independent of protein kinase C activation

March 1996

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The present study examines the effect of 1,2-dioctanoyl-sn-glycerol (DiC8), a diacylglycerol analogue, on L-type Ca2+ current (ICa,L) in adult rat ventricular myocytes using whole cell patch-clamp techniques. Extracellular application of DiC8 (1-10 microM) resulted in a concentration-dependent inhibition of peak ICa,L (half-maximum inhibitory concentration = 2.2 microM). Results obtained from the current-voltage relationship showed that DiC8 decreased the slope conductance. In addition, DiC8 increased the rate of Ba2+ current inactivation and caused a hyperpolarizing shift in the steady-state inactivation by 6 mV and a decrease in the slope factor. The DiC8-induced inhibition of ICa,L was neither mimicked by activation of protein kinase C (PKC) with 100 nM phorbol 12-myristate 13-acetate (PMA) no prevented by inhibition of PKC with 30 microM H-7, 100 nM staurosporine, or 24-h pretreatment with PMA. These results suggest that in rat ventricular myocytes 1) 1,2-sn-diacylglycerol (DAG) inhibits ICa,L, possibly by facilitating channel inactivation and decreasing channel availability and 2) this inhibitory effect of DAG is independent of PKC activation.

Effect of 1,2,4-triazole ethylamine on gastric secretion

April 1960

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8 Reads

3-(beta-aminoethylamine)-1,2,4-triazole, the triazole analogue of histamine, was found to be a potent excitant of gastric secretion, 0.42 mg of the triazole base being equivalent to 0.3 mg histamine in the dog. The action was slightly more prolonged than that of histamine. The evidence in this study along with earlier evidence strongly indicates that aminoguanidine (AG) acts additively with histamine, since AG inhibits the destruction of histamine. It was found that AG also augments the action of the triazole on gastric secretion, the mechanism of which is now uncertain.

Effects of 3-Amino-1,2,4-Triazole (AT) on Catalase and Other Compounds

August 1956

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19 Reads

Rat liver and kidney, but not blood, catalase activity decreases profoundly within the first 3 hours after the intraperitoneal or intravenous injection of AT. AT administered orally to mice causes a reduction of liver catalase activity. The liver and kidney catalase activity of rats returns to normal about 7 days after a single intraperitoneal injection. Liver cytochrome c content, hemoglobin level and urinary urobilinogen excretion are not affected by AT administration. Liver peroxidase activity is decreased slightly 3 hours after injection of AT but returns to normal within 24 hours. Prolonged AT administration has no effect on the growth rate of young rats. AT reduces the catalase activity of plant tissue homogenates, liver homogenates and crystalline catalase in vitro but only at high concentrations. AT causes a reduction of chlorophyll content and catalase activity of plants when administered in vivo but the relative effect against these two constituents varies with species, physiological state and concentration.

Role of 25-hydroxyvitamin D3 dose in determining rat 1,25-dihydroxyvitamin D3 production

June 1990

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6 Reads

To understand the relationships among 1) the dose of 25-hydroxyvitamin D [25(OH)D] in vivo, 2) the activity of 1-hydroxylase in renal mitochondria, and 3) the production of 1,25-dihydroxyvitamin D [1,25(OH)2D] in vivo, we gave rats different chronic or acute doses of 25-hydroxyvitamin D3 [25(OH)D3]. We followed the metabolism of intracardially administered [25-hydroxy-26,27-methyl-3H]cholecalciferol [25(OH)[3H]D3] for 24 h before killing by measuring extracts of serum by chromatography. Specific activity of 1-hydroxylase in kidney was measured at death. In rats given 0-2,000 pmol 25(OH)D3 chronically by mouth, there was a dose-dependent decline in the percent of serum radioactivity made up of 1,25-dihydroxy-[26,27-methyl-3H]cholecalciferol [1,25(OH)2[3H]D3] as well as a decline in mitochondrial 1-hydroxylase, and these correlated significantly (r = 0.83, P less than 0.001). Serum %1,25(OH)2[3H]D3 in this experiment ranged from 0.8 to 42%. A small part of this range could be accounted for by a faster metabolic clearance rate (MCR) of 1,25(OH)2D3 from rats supplemented with 25(OH)D3 (MCR, 2.12 +/- 0.10 ml/min) compared with rats restricted in vitamin D (MCR, 0.94 +/- 0.06 ml/min, P less than 0.001). The activity of 1-hydroxylase was by far the major factor determining serum %1,25(OH)2[3H]D3. When different acute doses of 25(OH)D3 were given to rats with identical specific activities of 1-hydroxylase, the resulting 1,25(OH)2D3 concentrations in serum correlated with the 25(OH)D3 dose (r = 0.99, P less than 0.001). We conclude that the behavior of 1-hydroxylase in vivo is analogous to the classic behavior in vitro of an enzyme functioning below its Michaelis constant (Km). The amount of 1-hydroxylase present in renal mitochondria determines the fraction (not simply the quantity) of 25(OH)D metabolized to 1,25(OH)2D3 in vivo.

Immunohistochemical localization of the 1,25(OH)2D3 receptor and calbindin D28k in human and rat pancreas

October 1994

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67 Reads

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is required for normal glucose-stimulated insulin release from pancreatic beta-cells. Biochemical characterization techniques have demonstrated the presence of the 1,25(OH)2D3 receptor (VDR) in homogenates of whole pancreas. Autoradiographic studies using radiolabeled 1,25(OH)2D3 suggest that the VDR is localized to beta-cells but are inconclusive. We used immunohistochemical techniques to stain serial sections from both human and rat pancreas with polyclonal antibodies to human VDR, chick calbindin D28k, insulin, glucagon, and somatostatin. VDR was present in the islet cells and also at low levels in acinar cells of the human and rat pancreas. Calbindin D28k was distributed in a manner similar to the VDR in pancreatic islets but was not present in acini. These results show for the first time that VDR and calbindin D28k are present in human pancreatic tissue. VDR and calbindin D28k are focally distributed throughout pancreatic islet cell types in humans and rats; VDR is also present in the exocrine pancreas. These findings suggest that 1,25(OH)2D3 may influence both endocrine and exocrine pancreatic function.

Glucocorticoids and appearance of 1,25-dihydroxyvitamin D3 receptor in rat intestine

April 1983

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8 Reads

The ontogenesis of the 1,25-dihydroxyvitamin D3 specific binding activity in intestine was examined in vitamin D-deficient and replete rats. The absence of binding activity in intestines during the first two postnatal weeks was not influenced by vitamin D supplementation. The concentration of binding sites peaked on day 18 in vitamin D-replete rats and preceded that in the deficient group by approximately 1 wk. The influence of glucocorticoids on 1,25-dihydroxyvitamin D3-binding protein levels was examined by sequential hydrocortisone administration and adrenalectomy. Subcutaneous hydrocortisone administration before day 14 postpartum did not induce binding activity. The concentration of binding sites was significantly increased to 369 +/- 60 fmol/mg of protein by hydrocortisone injections from days 15 to 17 postpartum when compared with an average of 182 +/- 16 fmol/mg of protein in littermate controls. Hydrocortisone administration did not further increase receptor levels in rats injected from days 19 to 21. Bilateral adrenalectomy on day 17 postpartum significantly decreased the concentration of binding sites. It is concluded that adrenal glucocorticoids play an important role in the developmental appearance of 1,25-dihydroxyvitamin D3 specific binding activity in the postnatal rat intestine.

Rapid nongenomic inhibition of renal 25-hydroxyvitamin D3 1-hydroxylase by 1,25-dihydroxyvitamin D3

September 1990

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4 Reads

The physiological role of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in inhibition of its own formation remains obscure. This study utilizes a kidney slice system to study the effect of physiological concentrations of 1,25(OH)2D3 on 25-hydroxyvitamin D3 1-hydroxylase (1-hydroxylase) formation in vitamin D-replete rats fed a normal-phosphate (NP) or low-phosphate (LP) diet. In vitro, 1-hydroxylase activity was assessed by measuring 1,25(OH)2D3 accumulation at 30 or 60 min after addition of 25-hydroxyvitamin D3 substrate. Degradation of 1,25(OH)2D3 was also assessed over 60 min. Rats fed the LP diet had a threefold increase in 1-hydroxylase activity but the same rate of degradation of 1,25(OH)2D3 as those fed the NP diet. The addition of 50 pM 1,25(OH)2D3 caused a proportional inhibition of 1-hydroxylase in NP and LP rats when added before or 10 min after addition of substrate but not at later time points; 150 pM 1,25(OH)2D3 completely inhibited 1-hydroxylase in the NP but not the LP rats. This inhibitory effect was not reversed by actinomycin D or cycloheximide. These results indicate that physiological concentrations of 1,25(OH)2D3 directly and rapidly modulate 1-hydroxylase activity via a nongenomic mechanism in both LP and NP diet rats.

Response of serum 1,25(OH)2D3 to variation of ionized calcium during chronic acidosis

October 1985

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10 Reads

Chronic ammonium chloride (NH4Cl) administration causes metabolic acidosis and prevents the normal rise of serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] during a low-calcium diet (LCD, 0.002% calcium). The suppression of serum 1,25(OH)2D3 was not due to reduced parathyroid hormone concentration, elevated serum phosphorus, or total calcium concentration. Acidosis increased blood ionized Ca [Ca2+] and proton [H+] concentrations. Serum 1,25(OH)2D3 levels were inversely correlated with both [Ca2+] and [H+]. To determine the independent effects of [Ca2+] on serum 1,25(OH)2D3 we varied [Ca2+] at a constant [H+] by infusing either EGTA or saline for 24 h after 11 days of LCD and NH4Cl. EGTA, preequilibrated with three concentrations of Ca, lowered [Ca2+] and raised 1,25(OH)2D3 but did not alter [H+] or serum phosphorus concentration. The log of serum 1,25(OH)2D3 varied linearly and inversely with arterial blood [Ca2+] during saline (r = -0.884, n = 8, P less than 0.001) and EGTA infusions (r = -0.798, n = 22, P less than 0.001). At all levels of [Ca2+], rats infused with EGTA had a higher serum 1,25(OH)2D3 than those infused with saline. Log serum 1,25(OH)2D3 was correlated neither with [H+] nor pH. Elevated [Ca2+] and not [H+] appears to suppress the serum 1,25(OH)2D3 response to LCD during NH4Cl acidosis in the rat.

Apparent [3H]1,25-dihydroxyvitamin D3 uptake by canine and rodent brain

April 1983

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16 Reads

The brain uptake of [3H]1,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3) was studied during steady-state conditions using the multiple-indicator dilution technique in dogs. The fractional [3H]1,25(OH)2D3 uptake was evaluated at 0.8 +/- 0.15% during a single passage through the dog brain. Evaluation of the [3H]1,25(OH)2D3 uptake by the vitamin D-replete and vitamin D-depleted rat brain indicated that 30 min after its injection, the fractional uptake was not influenced by the vitamin D status of the animals or by the amount of [3H]-1,25(OH)2D3 injected. In the rodent the fractional [3H]-1,25(OH)2D3 brain accumulation was between 0.16 and 0.20%, whereas the brain-to-serum ratio varied between 5 and 6%. Protein-binding studies of serum [3H]1,25(OH)2D3 indicated that, at 37 degrees C, 94.8 +/- 0.4% of the hormone was protein bound 30 min after its intravenous injection. These observations suggest that 1,25(OH)2D3 is able to cross the blood-brain barrier. However, its limited brain uptake in relation to its serum concentration suggests that the hormone does not penetrate freely into the central nervous system and that its brain uptake may be related to the free circulating 1,25(OH)2D3 concentration perfusing the blood-brain barrier.

Skeletal unloading decreases production of 1,25-dihydroxyvitamin D

May 1993

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6 Reads

The plasma concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] decreases during skeletal unloading and increases when normal weight bearing is restored. To determine whether these changes in plasma 1,25(OH)2D reflect changes in production, metabolic clearance, or both we measured the kinetics of 1,25(OH)2D metabolism in rats whose skeletons were normally loaded, unloaded, or reloaded after a period of nonweight bearing. Skeletal unloading produced a transient but striking fall in the production (-73%) and plasma concentration (-72%) of 1,25(OH)2D without having a significant effect (< 20%) on metabolic clearance. Skeletal reloading returned production to normal. Bone formation predictably decreased during unloading and returned to normal after return to weight bearing. No consistent changes in blood ionized calcium, plasma immunoreactive parathyroid hormone (irPTH), or plasma phosphorus occurred. These data suggest that the changes in plasma 1,25-(OH)2D associated with changes in skeletal weight bearing primarily reflect changes in 1,25(OH)2D production. The data provide no evidence that the changes in 1,25(OH)2D production are a consequence of changes in blood ionized calcium, plasma irPTH, or phosphorus.

1,25-Dihydroxyvitamin D3 inhibits Na+-dependent phosphate transport in osteoblastic cells

February 1993

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7 Reads

In the present work we investigated the influence of vitamin D3 metabolites on Na(+)-dependent phosphate (Pi) transport in the clonal osteoblastic cell line UMR-106. The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] dose-dependently inhibited Pi transport with a half-maximal concentration of approximately 5 x 10(-11) M. The effect of 1,25(OH)2D3 was first observed after 8 h of preincubation period. Inhibition of phosphate uptake was relatively specific for the 1,25(OH)2D3 analogue of vitamin D3. The potency order was 1,25(OH)2D3 > 24,25-dihydroxyvitamin D3 > 25-[3H]hydroxyvitamin D3. Kinetically, 1,25(OH)2D3 decreased the maximal velocity of the phosphate uptake system, whereas the affinity for phosphate was unaffected. Activation of protein kinase C (PKC) in UMR-106 cells stimulated Na(+)-dependent Pi transport. Nonetheless, the inhibitory effect of 1,25(OH)2D3 on Pi transport was not related to downregulation of PKC. Chemical determination of intracellular Pi showed a 50% reduction after 24-h preincubation with 10(-8) M 1,25(OH)2D3. We conclude that 1,25(OH)2D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells. This in turn leads to intracellular Pi depletion. The physiological implication of this phenomenon on the effects of vitamin D on osteoblasts in situ is discussed.

1,25(OH)2D3 binding along the rat nephron: autoradiographic study in isolated tubular segments

March 1985

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6 Reads

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding sites were studied along the nephron of rats. The animals were pretreated with the diphosphonate EHDP at doses that inhibit the endogenous production of 1,25(OH)2D3. A dry film autoradiographic technique was applied to tubular segments isolated by microdissection from kidneys incubated in vitro with various concentrations (0.2-12 nM) of [3H]1,25(OH)2D3 in the presence or absence of an excess unlabeled hormone (X200) in order to determine specific binding. Total, nonspecific, and specific labeling were quantified by silver grain counting over cytoplasmic and nuclear areas. Specific nuclear labeling appeared in the cortical ascending limb and papillary collecting tubule at 1 nM. In the distal tubule and, to a lesser extent, in the cortical collecting tubule a specific nuclear labeling was also present, but only at higher concentrations. No specific nuclear labeling was detected in the proximal tubule. All along the nephron, a significant and nonspecific labeling was observed in the cytoplasm, either alone or superimposed over the specific nuclear labeling. In conclusion 1,25(OH)2D3 specific binding sites appear to be localized mainly in the cortical ascending limb of the loop of Henle, in the distal and cortical collecting duct, and in the papillary collecting duct.

1,25-Dihydroxyvitamin D3-inducible catabolism of vitamin D metabolites in mouse intestine

May 1990

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5 Reads

The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-inducible C-24 oxidation pathway is a major catabolic pathway for vitamin D metabolites in target tissues. Using intestinal homogenates derived from 1,25(OH)2D3-treated mice, we examined the time course of induction, the intestinal localization and kinetics of induced enzyme activity, as well as the sensitivity of induction to transcriptional inhibitors actinomycin D and alpha-amanitin. 24-Hydroxylation of 500 nM 3H-labeled 25-hydroxyvitamin D3 [25(OH)D3] and 50 nM 3H-labeled 1,25(OH)2D3 by duodenal homogenates was detected 1 h after 1,25(OH)2D3 treatment; C-24 oxidation products of 25(OH)D3 and 1,25(OH)2D3 peaked at approximately 6 h and remained elevated for 17 h. Induced enzyme activity was localized to the mitochondrial fraction, was highest in duodenum, and was also detected in jejunum, ileum, and colon. The apparent Michaelis constant of the induced duodenal enzyme for 25(OH)D3 was 451 nM and for 1,25(OH)2D3 was 14 nM. Induction of intestinal catabolic activity was inhibited by prior treatment of 1,25(OH)2D3-injected mice with either actinomycin D or alpha-amanitin. The characteristics of the 1,25(OH)2D3-inducible C-24 oxidation pathway in the intestine resembled that of the kidney. However, the catabolic pathway was constitutively expressed only in the kidney. We conclude that 1,25(OH)2D3-inducible degradation of vitamin D metabolites occurs throughout the length of mouse intestine and can be prevented by transcriptional inhibitors, suggesting that mRNA synthesis is required for the induction process.

Active Ca2+ transport in primary cultures of rabbit kidney CCD: Stimulation by 1,25-dihydroxyvitamin D3 and PTH

December 1991

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11 Reads

Rabbit connecting tubules and cortical collecting ducts, which represent 79 +/- 5% of the calbindin-D28k-containing kidney cells, were isolated by immunodissection from the rabbit kidney superficial cortex and seeded on permeable filters. After 6 days in culture the monolayers developed a potential difference (PD) of -24 +/- 3 mV (lumen negative) and a transepithelial resistance (R) of 284 +/- 19 omega.cm2. Addition of 10(-6) M amiloride to or removal of Na+ from the mucosal side reversed the PD to +6 +/- 4 mV and concomitantly increased R to 660 +/- 122 omega.cm2. The cells developed functional parathyroid hormone (PTH) and arginine vasopressin receptors, but calcitonin receptors were absent. The monolayer actively absorbed Ca2+ against an electrochemical gradient with a rate of 121 +/- 13 nmol.h-1.cm-2. Removal of serosal Na+ inhibited Ca2+ absorption by 63 +/- 8%. Exposure to 10(-7) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for 48 h or 10(-7) M bovine PTH (bPTH)-(1-34) for 1 h, increased transcellular Ca2+ absorption by 53 +/- 13% or 24 +/- 8%, respectively. The effects of 1,25(OH)2D3 and PTH in combination were neither additive nor potentiating. In addition, the cultured cells expressed calbindin-D28k, and, after exposure to 10(-7) M 1,25(OH)2D3 for 48 h, the calbindin-D28k content increased fourfold.

Interactions between retinoic acid and 1,25(OH)2D in mouse immortalized osteoblastic C1 cells

June 1994

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3 Reads

The interactions between 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and retinoic acid (RA) on alkaline phosphatase (ALP) activity and vitamin D receptors (VDR) were determined in the SV40 immortalized osteoblastic cell line C1. Biochemical and cytochemical analysis showed that ALP activity increased with C1 cell density and that 1,25(OH)2D inhibited ALP activity at high density, whereas RA increased ALP activity at low density. The interactions of the two hormones were also cell density dependent, with a predominant stimulatory effect of RA at low density and a predominant inhibitory effect of 1,25(OH)2D at high density. In contrast, the two hormones inhibited C1 cell proliferation independently of cell density. Scatchard analysis and immunocytochemical staining showed that nuclear VDR levels increased with cell density and that RA, 1,25(OH)2D, and their combination upregulated VDR levels mainly at high cell density. Although RA increased VDR levels, RA did not potentiate the effect of 1,25(OH)2D on ALP activity. The results indicate that the effects and interactions of 1,25(OH)2D and RA on ALP activity in osteoblastic C1 cells depend on the state of phenotypic maturation.

TABLE 2 . Vitamin D3 metabolites detected by high-performance liquid chromatography 
Intestinal metabolism and portal venous transport of 1,25(OH)2D3, 25(OH)D3, and vitamin D3 in the rat
  • Article
  • Full-text available

July 1985

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91 Reads

We compared the intestinal absorption of three vitamin D3 sterols and tested the hypothesis that the intestine hydroxylates absorbed vitamin D and transports polar metabolites in portal venous blood. Micellar solutions containing 50 nmol of a radiolabeled vitamin D3 sterol (1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or vitamin D3) were placed in closed jejunal segments of rats prepared with lymphatic and mesenteric venous fistulas. Venous blood loss was replaced by infusion of donor rat blood into the saphenous vein. After 1-2 h the rats were killed, and intestinal homogenates, mesenteric blood, and lymph were analyzed. The average rate of absorption of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was two- and fivefold higher than that of 25-hydroxyvitamin D3 [25(OH)D3] and vitamin D3 (D3), respectively. Transport of hydroxylated vitamin D sterols was primarily via the venous route; the average rate of venous transport of 1,25(OH)2D3 was 18.3 X 10(2) nmol X min-1 X g-1 compared with 8.8 X 10(2) for 25(OH)D3 and 0.13 X 10(2) for D3. High-performance liquid chromatography of intestinal and plasma extracts revealed that there was 25-hydroxylation of absorbed D3, 24- and putative 1-hydroxylation of absorbed 25(OH)D3, and prompt portal venous transport of all hydroxylated metabolites. When 1,25(OH)2D3 was infused into the lumen, the composition of radiolabeled sterols found in intestinal homogenates and mesenteric venous plasma was virtually identical to that of the infusate. These studies provide in vivo evidence for the intestinal metabolism of pharmacological quantities of absorbed vitamin D3 sterols and the prompt portal venous transport of more polar metabolites.
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Effect of 1,25 dihydroxyvitamin D3 on isolated islets from vitamin D3-deprived rats

May 1990

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8 Reads

Insulin release is impaired by vitamin D3 deficiency but can be restored by in vivo administration of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. A direct influence of 1,25(OH)2D3 on the B-cell was studied in vitro with islets from 5-wk vitamin D3-deprived rats. This hormone (10(-12) to 10(-6) mol/l) added to the incubation medium had a stimulatory dose-dependent effect on insulin response to 8.3 mmol/l glucose 6 h later. Moreover, perifusion experiments performed after different times of incubation demonstrated that after 6 h 1,25(OH)2D3 increased in particular the first phase of insulin response to 16.7 mmol/l glucose. The 45Ca fluxes, followed in parallel, were never modified by 1,25(OH)2D3 in the absence of glucose but were enhanced during the glucose stimulus, whereas 86Rb fluxes were never affected by 1,25(OH)2D3. These results demonstrated that 1,25(OH)2D3 acts in vitro on B-cells, but with a 6-h delay to potentiate the glucose-induced insulin release, concomitant with intracellular calcium redistribution.

1,25-Dihydroxyvitamin D3 restores fertility of vitamin D-deficient female rats

May 1989

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212 Reads

Vitamin D deficiency reduces mating success and fertility in female rats, but it is not known if the reduction in reproductive performance is a direct action of vitamin D or the hypocalcemia associated with vitamin D deficiency. The effect of vitamin D deficiency with normocalcemia on fertility and reproductive capacity in female rats was investigated. Female weanling rats were maintained on vitamin D-deficient or vitamin D-replete diets until maturity and mated to age-matched, normal, vitamin D-replete males. Three groups of vitamin D-deficient females were maintained on diets varying in calcium and Pi concentrations to test the effect of vitamin D deficiency with different serum calcium and Pi concentrations on reproductive performance. Vitamin D-deficient females were capable of reproduction, but successful matings by all groups of vitamin D-deficient females were markedly reduced regardless of serum calcium concentration, when compared with matings with vitamin D-replete females. Fertility was also drastically reduced in litters from all groups of vitamin D-deficient females regardless of serum calcium concentration, when compared with litters from vitamin D-replete females. Vitamin D-deficient female rats that received vitamin D or 1,25-dihydroxyvitamin D3 were capable of successfully mating and giving rise to normal, healthy litters. These results indicate that vitamin D and not hypocalcemia is directly responsible for reduced reproductive capacity and fertility in vitamin D-deficient female rats.

Low-calcium diets increase both production and clearance of 1,25-dihydroxyvitamin D3 in rats

March 1990

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8 Reads

Administration of large doses of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to animals induces 1,25(OH)2D3 side-chain oxidative pathways. This study determined if the elevated plasma 1,25(OH)2D3 seen in rats fed low-Ca diets is associated not only with an increased production rate (PR) but also with an increased metabolic clearance rate (MCR) of the hormone. In vitamin D-replete rats fed a Ca-deficient diet for 3-4 wk, the PR increased 21-fold, plasma levels 15-fold, and the MCR by 37%. The increased MCR in Ca-deficient rats was associated with a 48% increase in hepatic microsomal UDP glucuronyl transferase enzyme activity, whereas 1,25(OH)2D3 catabolism by homogenates of liver and small intestinal mucosa was unchanged. In contrast to the effects of low-Ca diets, acute (7 h) pharmacological elevation of plasma 1,25(OH)2D3 to 1.5 ng/ml in normal rats did not influence the MCR. Thus chronically elevated 1,25(OH)2D3 levels are necessary to stimulate clearance. In conclusion, 1,25(OH)2D3 clearance in rats can be stimulated not only by chronic pharmacological doses of 1,25(OH)2D3 but also by the physiological stimulus of a low-Ca diet. Hence, plasma 1,25(OH)2D3 levels can be regulated by changes in both PR and MCR.

Effects of chlorothiazide on 1,25-dihydroxyvitamin D3, parathyroid hormone, and intestinal calcium absorption in the rat

July 1982

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14 Reads

Previous studies have shown that thiazide diuretic agents reverse secondary hyperparathyroidism and reduce circulating 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and intestinal calcium absorption rates in patients with idiopathic hypercalciuria of the renal-leak variety. We have investigated whether thiazides can reverse the secondary increase in serum parathyroid hormone (PTH) and 1,25(OH)2D3 levels or intestinal calcium absorption induced by feeding rats a diet low in calcium (LCD, 0.02% calcium) but adequate in phosphorus and vitamin D. We found that LCD increased circulating immunoreactive PTH [chow vs. LCD, 0.52 +/- 0.06 vs. 1.06 +2- 0.1 (SE) ng/ml, P less than 0.001], 1,25(OH)2D3 (chow vs. LCD, 101 +/- 15 vs. 325 +/- 38 pg/ml, P less than 0.001), calcium uptake by everted gut sacs from duodenum, ileum, and descending colon, and net calcium absorption by descending colon studied in Ussing chambers in vitro. Chlorothiazide (CTZ) prevented the increase in PTH during LCD (chow + CTZ vs. LCD + CTZ, 0.69 +/- 0.07 vs. 0.73 +/- 0.06, NS) but not the increase in 1,25(OH)2D3 (chow + CTZ vs. LCD + CTZ, 88 +/- 10 vs. 277 +/- 31, P less than 0.002) or intestinal calcium transport. The drug caused no change in serum 1,25(OH)2D3 or intestinal calcium absorption in rats fed normal chow. In rats given exogenous 1,25(OH)2D3 to stimulate intestinal calcium absorption, CTZ reduced urine calcium excretion greatly but did not alter intestinal calcium absorption.

Parathyroid hormone-independent regulation of 1,25(OH)2D in response to inhibition of bone resorption

April 1988

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16 Reads

Both plasma level of 1,25-dihydroxyvitamin D [1,25(OH)2D] and intestinal Ca absorption increase after biphosphonate-induced inhibition of bone resorption. Parathyroid hormone (PTH) has been considered a key mediating element of this homeostatic response. In the present work, the role of PTH was assessed by studying the influence of 1-hydroxypentane-1,1-bisphosphonate (HPeBP) on vitamin D and Ca metabolism in both intact and thyroparathyroidectomized (TPTX) rats. In intact rats, HPeBP given at 0.1 mg P/kg body wt sc for 10 days strongly inhibited bone resorption without affecting bone formation. This effect was associated with a marked stimulation of intestinal Ca absorption and Ca balance. In this condition, HPeBP caused a marked rise in plasma 1.25(OH)2D without affecting the level of 25-hydroxyvitamin D. In TPTX rats, HPeBP given at same dose also inhibited bone resorption and enhanced plasma 1,25(OH)2D, intestinal Ca absorption and Ca balance. In summary, this study shows that bisphosphonates such as HPeBP with prevailing inhibitory activity on bone resorption induce a marked stimulation of both 1,25(OH)2D production and intestinal Ca absorption. This homeostatic response is not attenuated after PTH removal. Thus, as previously shown for the response to low Ca diet, PTH does not appear to be an essential mediating factor for stimulating 1,25(OH)2D production in response to an increase in bone mineral retention.

Postnatal changes in plasma 1,25-dihydroxyvitamin D3 in sheep: Role of altered clearance

December 1991

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2 Reads

The ontogeny of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production and metabolic clearance rates (PR and MCR, respectively) was determined in chronically catheterized fetal (postconceptional age of 127 days; term = 145 days), neonatal (10 +/- 1 and again at 24 +/- 1 days postnatal), and adult (3 yr of age) sheep. Plasma concentrations of 1,25(OH)2D3 were more than twofold higher in fetuses than in adults and were increased by 78% during the 1st 24 days postnatally. In contrast, compared with values in fetuses, the MCR of 1,25(OH)2D3 was reduced by 40% in 10-day-old neonates and by 58% in 24-day neonates, although 24-day neonatal values were still 75% higher than adult values. In contrast, the PR of 1,25(OH)2D3 did not demonstrate an ontogenic change during development and remained at all times approximately ninefold the adult value. The finding, by regression analysis of an inverse relationship between the MCR and the plasma concentration of 1,25(OH)2D3, is interpreted to suggest that increased plasma concentrations are the result of a reduced MCR. We conclude that increases in plasma concentrations of 1,25(OH)2D3 in the perinatal period can occur by a mechanism that is independent of enhanced endogenous synthesis, namely a reduction in the MCR of the hormone.

Different responses of trabecular and cortical bone to 1,25(OH)2D3 infusion

December 1990

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7 Reads

Previous studies regarding the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone have suggested that 1,25(OH)2D3 increases bone mass and calcium. Many of these studies have focused on trabecular or total bone without examining cortical bone per se. To determine whether the response of trabecular bone to 1,25(OH)2D3 differed from the response of cortical bone, we infused 1,25(OH)2D3 into rats and examined bone mass, 45Ca accumulation, and the density distribution of bone particles (as a measure of bone maturation) in both the proximal tibia and shaft. In the proximal tibia 1,25(OH)2D3 decreased 45Ca accumulation, yet increased bone mass and shifted the particle distribution to more mineralized fractions. In the shaft there was a redistribution of bone to less mineralized fractions that was not accompanied by a change in total bone mass or a decrease in 45Ca accumulation. Thus 1,25(OH)2D3 may retard bone maturation and mineralization throughout the tibia, but this effect in the proximal tibia appears to be overshadowed by a reduction in bone resorption resulting in an accumulation of well-mineralized bone in that region. Bone resorption, however, was not measured directly. The net result is an increase in bone mass and density of trabecular bone not seen in cortical bone.

Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in Caco-2 cells

May 1999

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21 Reads

Calcium transport in the apical-to-basolateral (A-to-B) or B-to-A direction was examined in cells treated with 10 nM 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3, calcitriol] for up to 72 h. Net A-to-B calcium transport was positive at all time points and increased from 0.14 +/- 0.06 to 0.50 +/- 0.01 nmol. well-1. min-1 after 72 h of calcitriol treatment. Neither phenol red transport nor transepithelial electrical resistance was altered by calcitriol treatment, suggesting that the increase in net A-to-B calcium transport was not due to paracellular movement. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 (100 nM, 48 h) alters basal or calcitriol-stimulated A-to-B calcium transport. Treatment with the calmodulin antagonist trifluoperazine (50 microM) reduced calcitriol-stimulated A-to-B Ca transport by 56%. The transcription inhibitor actinomycin D inhibited calcitriol-regulated A-to-B calcium transport as well as calbindin D9k and 24-hydroxylase mRNA accumulation. These data demonstrate that calcitriol-mediated A-to-B calcium transport in Caco-2 cells is a specific, transcellular process that requires transcriptional events normally mediated through the vitamin D receptor.

Elevated 1,25-dihydroxyvitamin D3 and intestinal calbindin-D9k in the toothless rat

March 1990

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3 Reads

The toothless (tl) rat is a nonlethal osteopetrotic mutation characterized by systemic skeletal sclerosis, growth plate morphology suggestive of rickets, and morphological evidence of reduced osteoclastic bone resorption. Vitamin D metabolites, serum calcium and phosphorus levels, and the developmental appearance of vitamin D-dependent intestinal calcium binding protein (calbindin-D9k) was studied in normal and mutant rats of tl stock from 7 to 35 days of age. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was found to be significantly elevated in mutant animals by 7 days of age (71 +/- 9 pM, tl/tl vs. 24 +/- 8 pM, +/?) and continued to increase to a peak of 428 pM at the time of weaning. This was 240% higher than normals at this period. The elevated levels of 1,25-(OH)2D3 stimulated a significant and precocious appearance of intestinal calbindin-D9k in mutants, beginning by 14 days of age and reaching their peak levels at 21 days postpartum (25.6 +/- 1.7 micrograms/mg protein, tl/tl vs. 16.4 +/- 1.5 micrograms/mg protein, +/?). The cause of the elevated circulating levels of 1,25-(OH)2D3 in tl rats is unknown but may be due to the low serum phosphorus levels present in these animals.

Metabolic acidosis reverses the increase in serum 1,25(OH)2D in phosphorus-restricted normal men

December 1992

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20 Reads

We tested the hypothesis that in humans, metabolic acidosis can disorder the metabolism of 1,25-dihydroxyvitamin D [1,25(OH)2D] by impairing the capacity for a sustained physiological stimulus to increase renal production of this hormone. Specifically, in seven healthy men in whom restriction of dietary phosphorus had doubled their serum concentration of 1,25(OH)2D, we induced metabolic acidosis of moderate severity with oral NH4Cl, administered for 7 days. With induction of acidosis, the serum concentration of 1,25(OH)2D decreased sharply and remained decreased and near constant throughout the period of acidosis, the decrease amounting to one-half of the increment induced by phosphorus restriction alone. The serum concentration of free 1,25(OH)2D also decreased, since the measured free fraction of 1,25(OH)2D was unaffected by NH4Cl. The decrease in serum 1,25(OH)2D was accounted for by a 16% increase in its metabolic clearance rate and by a 19% decrease in its production rate. Metabolic acidosis induced a modest increase in the concentrations of blood ionized calcium and serum phosphorus. Multiple linear regression analysis revealed that serum levels of 1,25(OH)2D varied inversely and significantly with those of plasma hydrogen ion (R = -0.77, P < 0.001), but not with those of blood ionized calcium or serum phosphorus. These data demonstrate in humans that metabolic acidosis can substantially reverse the increase in serum concentration of 1,25(OH)2D induced by phosphorus restriction. The data provide evidence that acidosis can restrict the increase in renal production and serum concentration of 1,25(OH)2D effected by a sustained physiological stimulus.

Increased clearance of 1,25(OH)2D3 and tissue-specific responsiveness to 1,25(OH)2D3 in diabetic rats

September 1993

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The kinetics of 1,25-dihydroxyvitamin D3 [1,25(OH)2-D3] and the in vivo response to 1,25(OH)2D3 (7.5, 15, and 30 ng/100 g body wt), infused or injected subcutaneously for 12-14 days, were studied in male spontaneously diabetic and control BB rats. In control rats, increasing doses of 1,25(OH)2D3 produced parallel increases in plasma 1,25(OH)2D3 and calcium, urinary calcium, duodenal CaBP9K, and renal CaBP28K. 1,25-(OH)2D3 at 30 ng/100 g markedly raised plasma osteocalcin and osteoblast/osteoid surfaces in the tibial metaphysis, but inhibited bone mineralization rate. In diabetic rats, plasma 1,25-(OH)2D3 concentrations were decreased, and the rise of plasma 1,25(OH)2D3 during 1,25(OH)2D3 infusion was blunted, but the free 1,25(OH)2D3 index remained normal or above normal. Diabetic rats had an increased metabolic clearance rate of 1,25-(OH)2D3 (0.38 +/- 0.015 vs. 0.24 +/- 0.007 ml.min-1.kg-1), with no further increase in 1,25(OH)2D3-infused diabetic rats; their relative production rate of 1,25(OH)2D3 was unchanged. The responses of plasma and urinary calcium, duodenal CaBP9K, and renal CaBP28K to infused 1,25(OH)2D3 were normal, as was duodenal calcium absorption in 1,25(OH)2D3-injected diabetic rats. However, the virtual absence of osteoblasts/osteoid in trabecular bone was unaltered in diabetic rats infused with 30 ng/100 g 1,25(OH)2D3, with only minimal increase of their low plasma osteocalcin levels. 1,25(OH)2D3 treatment therefore cannot be expected to reverse diabetic osteopenia.

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