Cyclosporin A stimulates apical Na+/H+ exchange in LLC-PK1/PKE20 proximal tubular cells

Cyclosporin A (CyA) causes renal Na⁺ retention which may lead to arterial hypertension. The apical Na⁺/H⁺ exchanger (NHE3) is responsible for bulk proximal tubular Na⁺ reabsorption. The aim of this study was to investigate the effects of CyA on the NHE3 of polarized proximal tubular cells to evaluate cellular mechanisms of CyA-associated arterial hypertension. The change of the intracellular pH (Δ-[pH]_i/min) was determined as a measure of the activity of the NHE in LLC-PK₁/PKE₂₀ cells using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NHE activity was identified as the apical NHE3 since it could be inhibited by the inhibitor S3226, but not by inhibitors of the basolateral isoform (NHE1) amiloride or HOE 694. CyA stimulated the NHE3 activity dose dependently. The mean increase stimulated by relevant CyA concentrations was 61±11%. A 24-h application of CyA also stimulated an increase of NHE3 activity which did not seem to be mediated by an increase of NHE3 RNA expression. The less immunosuppressive derivatives cyclosporin H and cyclosporin G caused NHE3 activation as well. Carbachol and ATP, which both induce a Ca²⁺ release from internal Ca²⁺ stores, also increased the NHE3 activity. The Ca²⁺ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,-N′,N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) abolished the CyA-associated NHE3 stimulation, whereas low extracellular Ca²⁺ had no effect. CyA-associated effects did not seem to be mediated via inhibition of protein kinase C (PKC). CyA had no additive effects on the angiotensin II-associated NHE3 stimulation. Concurrent application of losartan did not impair the CyA-induced NHE3 stimulation. In conclusion CyA stimulates the apical NHE3 in proximal tubular cells. This is mediated by Ca²⁺ release from intracellular stores but is independent of the action of angiotensin II or PKC.     

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

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

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

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

Neurotransmitter regulation of cytosolic calcium in osteoblast-like bone cells

Summary The nervous system may play a role in regulation of bone metabolism. The effects of norepinephrine(NE), vasoactive intestinal peptide(VIP), and ATP on cytosolic Ca²⁺ were assessed in a rat osteoblast-like osteosarcoma cell line (UMR-106) responsive to PTH. All three transmitters transiently increased Ca²⁺, with ATP≫PTH>NE=VIP, and then caused sustained increases in Ca²⁺. The ATP-induced transient resulted from mobilization of intracellular Ca²⁺ store, while NE and VIP-induced transients also involved influx of Ca²⁺. Later sustained increases by all agonists were dependent upon extracellular Ca²⁺. Release of intracellular Ca²⁺ by ATP was associated with a marked increase in IP3 but without a significant change in cAMP. NE, VIP, and ATP, through regulation of Ca²⁺ metabolism, may be involved in various osteoporotic conditions.     

Variation of increases in free cytosolic calcium ion induced by prostaglandin E2 in mouse osteoblast clone,MC3T3-E1—Single cell assay using microspectro-fluorometry

Osteoblasts exhibit multiple phenotypic expression in response to prostaglandin E₂ (PGE₂). Intracellular calcium concentration ([Ca²⁺] _i ) was elevated by PGE₂ treatment in the mouse osteoblast clone, MC3T3-E1, but the degree of elevation was varied by the day after subculturing. To study the different response to PGE₂, we have used microspectrofluorometry to measure [Ca²⁺] _i in a single MC3T3-E1 cell loaded with fura-2. In the presence of extracellular Ca²⁺, the increase in [Ca²⁺] _i in the osteoblast exhibited multiple patterns. The patterns were roughly classified into four groups by the time reached maximum level; “transient”, “gradual”, “transient and gradual” and “no response”. Within 2 days after subculturing, the cells showing “gradual” and “no response” were predominant, whereas after day 3 the cells showing “transient” and “transient and gradual” were predominant. We also investigated the daily change in the maximum level of [Ca²⁺] _i in the cells showed “transient” in response to PGE₂. The magnitude of [Ca²⁺] _i increase was also varied in cultivating period. These data suggest that there are phenotypic variations in a single cell even in a cloned cell line and this phenotype may change in the stage of cell proliferation.     

Monoclonal antibodies against the putative divalent cation-receptor that is located on parathyroid cells do not stain isolated rat osteoclasts

Summary It has been reported that osteoclastic function is regulated by calcium-induced alterations in cytoplasmic free calcium ([Ca²⁺]_i), possibly through a specific receptor. We have investigated whether osteoclasts, isolated from neonatal rat long bones, possess the divalent cation-receptor that has been demonstrated on parathyroid cells. Studies with fura-2 loaded adherent single cells showed that an increase in extracellular Ca²⁺ ([Ca²⁺]_e) from 0.5 mM to 10 mM resulted in an increase in [Ca²⁺]_i in isolated rat osteoclasts, from a basal value of 94.7±16.2 to 150.6±22.4 nM (means±SEM; n=14). The shape and time course of the [Ca²⁺]_i increase varied considerably from cell to cell. Less than half of the cells responded with a rapid transient increase whereas the rest responded with a slow increase that reached a plateau within 1–2 minutes. When [Ca²⁺]_e was changed back to 0.5 mM, a slow decrease in [Ca²⁺]_i was monitored. Immunohistochemical staining with two different monoclonal antibodies, recognizing the putative Ca²⁺ receptor on parathyroid cells, did not indicate any staining on freshly isolated rat osteoclasts. Thus, our data demonstrate that an increase in [Ca²⁺]_e causes an elevation of [Ca²⁺]_i in osteoclasts. This increase is not mediated via the putative cation-receptor found on parathyroid cells.     

Effects of 1,25 dihydroxyvitamin D3 administration on circadian mineral rhythms in humans

Summary 1,25 Dihydroxyvitamin D₃ (1,25(OH)₂D₃) (2.0 μg) was given intramuscularly to 6 healthy adult males. Twenty-four circadian patterns of blood-ionized calcium (Ca²⁺), serum phosphate (P_i), and total calcium (Ca_T) were assessed pre- and posthormone administration. Correlations of mean mineral rhythms with normative models were significant for each mineral pattern on both study days. Mean Ca²⁺ and Ca_T rhythms became weakly correlated after hormone treatment (r=.39). A small but statistically significant increment in the 24 h grand mean Ca²⁺ concentration was observed on the treatment day compared with the baseline day. However, this increment is less than the year-to-year variability in the grand mean mineral concentrations derived from the same subjects studied under baseline conditions previously. These data indicate that acute parenteral administration of near-physiological (2.0 μg) doses of 1,25(OH)₂D₃ appears to have no major effect on circadian mineral pattern shape or mean mineral concentrations.     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.     

Inhibition by 1,25 dihydroxycholecalciferol of hormonal secretion of rat parathyroid gland in organ culture

Summary The effect of vitamin D metabolites on parathyroid hormone secretion was studied using rat parathyroid gland cultured in basal medium Eagle containing 5% serum obtained from thyroparathyroidectomized rat, 1 mM magnesium, and calcium concentration varying from 0.75–2.25 mM, and radioimmunoassay for rat parathyroid hormone (rPTH). 1,25 dihydroxycholecalciferol (1,25(OH)₂D₃), 5×10⁻¹⁰−2.5×10⁻⁸M, consistently decreased rPTH secretion in dose-related manner; the effect reached steady state after 24 hin vitro addition of 1,25(OH)₂D₃ and was also observed at different medium calcium concentrations (0.75, 1.25, 1.75 mM). Comparison of dose-responses for inhibitory activity of some vitamin D metabolites on rPTH secretion showed: 1,25(OH)₂D₃=1,24,25(OH)₃D₃>1α OHD₃>25 OHD₃. Cholecalciferol (10⁻⁵M), 24,25-dihydroxycholecalciferol (10⁻⁸−10⁻⁶M) and 25,26-dihydroxycholecalciferol (5×10⁻⁹−5×10⁻⁷M) did not inhibit rPTH secretion. Analysis of structural activity relation of vitamin D metabolites studied indicated that 1α or pseudo-1α hydroxylated metabolites or analogs were active in inhibiting rPTH secretion, while, non-1α hydroxylated metabolites were without or were weakly inhibitory only at very high concentrations. This study provides further evidence for a direct role of 1,25(OH)₂D₃ on a negative feedback loop for regulation of parathyroid gland function.     

Role of intracellular Ca2+ stores in smooth muscle contractions of the guinea pig vas deferens

Summary Guinea pig vas deferens was used as an animal model for alpha-1 adrenoceptor (₁-receptor) mediated contractions in human hyperplastic prostatic tissue. The selective ₁-receptor agonist, phenylephrine (PE), induced fully reversible, dose-dependent contractions antagonized by increasing concentrations of the ₁-receptor blockers prazosin (1–100 nM) and YM 617 (0.1–10 nM). Removal of extracellular Ca²⁺ reduced PE-evoked contractions in a time-dependent manner. Nifedipine (1–1000 nM), a blocker of voltage-dependent L-type Ca²⁺ channels (VDCC), inhibited the PE-induced response by up to 65%. Removal of extracellular Ca²⁺ abolished the ₁-agonist reactivity in a time-dependent fashion. To elucidate the participation of intracellular Ca²⁺ stores in ₁-receptor contractions, the tissue was pretreated with ryanodine (10 M) or thapsigargin (0.1 M), established inhibitors of Ca²⁺ release from intracellular pools. Both substances reduced the PE contractions by up to 80%. Nifedipine suppressed the remaining contractions completely. This provides evidence that Ca²⁺ influx through VDCC and Ca²⁺ release from intracellular stores contribute to ₁-receptor contractions in the guinea pig vas deferens and may be important in obstructive benign prostatic hyperplasia.     

In vitro calcium transport properties of skeletal muscle mitochondria from vitamin D-deficient and 1,25-dihydroxy-vitamin D3-treated chicks

Summary Previous work has shown that vitamin D₃ or 1,25-dihydroxy-vitamin D₃ affect calcium content and fluxes in mitochondria of chick skeletal musclein situ. Studies were performed to investigate whether these effects are related to variations in the Ca²⁺ transport properties of mitochondrial membranes. Mitochondria isolated from skeletal muscle of vitamin D-deficient chicks and chicks dosed with 1,25(OH)₂D₃ for 3 or 7 days (50 ng/day) were employed. No changes in the rate and affinity for calcium of the Ruthenium Red-sensitive Ca²⁺ uptake system were detected after treatment with 1,25(OH)₂D₃. The metabolite did not cause either modifications in Ca²⁺ efflux from mitochondria preloaded with the cation induced by Na⁺ or blockage of mitochondria energy supply. Prior treatment of animals with vitamin D₃ was also without effects. However, a significant stimulation of Ca²⁺ uptake by intact muscle preparations from the same experimental animals was observed in response to treatment with 1,25(OH)₂D₃ in vivo (50 ng/day, 3 days) orin vitro (10⁻¹⁰ M, 60 minutes). In addition, the Ca content of muscle mitochondria was markedly diminished in chicks treated with the sterol. It is suggested that the effects of 1,25(OH)₂D₃ on muscle mitochondrial Ca metabolism may be secondary to changes in cytoplasmic Ca²⁺.     

αVβ3 Integrin ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes

We propose that specific osteocyte–matrix interactions regulate the volume-sensitive calcium influx pathway, which we have shown is mediated by stretch-activated cation channels (SA-Cat) and is essential for the stretch-activated anabolic response in bone. The current study measured the hypotonic swelling-induced increase in cytosolic calcium concentration, [Ca²⁺]_i, in rat osteocytes, and found that cells adherent to different matrices behave differently. Osteopontin and vitronectin, matrix molecules that bind the α_Vβ₃ integrin, induced larger responses to the hypotonic swelling than other matrix molecules that bind other integrins. Addition of echistatin, which is a soluble α_Vβ₃ ligand, significantly enhanced the hypotonic [Ca²⁺]_i increase in addition to inducing an immediate increase in [Ca²⁺]_i by itself. These results strongly support the contention that α_Vβ₃ integrin signaling in osteocytes interacts with that in mechanotransduction, which is downstream of SA-Cat.     

In vitro evidence that local and systemic skeletal effectors can regulate3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract

Summary These investigations were intended to determine whether local and systemic skeletal effectors—3′5′-cyclic adenosine monophosphate (cAMP), prostaglandin E₂ (PGE₂), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)₂D), calcitonin, and NaF—could regulate³[H]-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase³[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 μM) and low-dose PTH (0.1 nM) stimulated³[H]-thymidine incorporation,P<.05 for each; isobutylmethylxanthine (IBMX-1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased³[H]-thymidine incorporation; and PGE₂ (1 μM) had no effect. The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)₂D were acute. PTH had complex time-dependent actions on³[H]-thymidine incorporation, being inhibitory after 4–8 hours of exposure and stimulatory after 20–24 hours (P<.001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase³[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited³[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P<.005). The inhibitory actions of IBMX and 1,25(OH)₂D were independent of cell differentiation. Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase³[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase³[H]-thymidine incorporation (P<.001 for each). These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on³[H]-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract. High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease³[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase³[H]-thymidine incorporation. Both IBMX and PGE₂ were competitive inhibitors of bone extract-stimulated³[H]-thymidine incorporation (P<.001 for each), implying that these effectors (IBMX, PGE₂, and embryonic chick bone extract) shared a common (or coincidentally equal) rate-limiting step. The effects of 1,25(OH)₂D on bone extract-stimulated³[H]-thymidine incorporation were different at high and low doses. At a low concentration (1 nM), 1,25(OH)₂D enhanced the effect of bone extract to increase³[H]-thymidine incorporation, but higher concentrations (e.g., 100 nM) were inhibitory (P<.01 for each). Together, these data demonstrate that local and systemic skeletal effectors can have direct effects on embryonic chick calvarial cells,in vitro, to regulate the basal rate of³[H]-thymidine incorporation, and to modulate the stimulatory action of an embryonic chick bone extract.     

Somatostatin inhibits duodenal calcium transport mediated by 1,25-dihydroxyvitamin D3 in perfused duodena from normal chicks

We have reported that physiological dose (30pM-650pM) of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] increased the unidirectional movement of⁴⁵Ca²⁺ from the lumen to the venous effluent within a few minutes in perfused duodena from normal chicks, and hypercalcemia inhibited this rapid stimulatory effect on calcium transport mediated by 1,25(OH)₂ D₃. The purpose of the present study was to determine the effect of somatostatin on calcium transport in chicks. The basal Ca²⁺ transport, in the absence of 1,25(OH)₂ D₃, did not change when 10⁻⁸M to 10⁻⁶M of somatostatin was added to the perfusate. The effect of 1,25(OH)₂D₃ on calcium transport, however, was completely abolished on addtion of 10⁻⁶M somatostatin in the perfusate, and partially blocked on addition of 10⁻⁷M somatostatin and 10⁻⁸M somatostatin had no effect on 1,25(OH)₂ D₃ mediated calcium transport. These results suggest that somatostatin may decrease intestinal calcium transport mediated by the rapid direct action of 1,25(OH)₂ D₃.     

Characteristics of spontaneous calcium oscillations in renal tubular epithelial cells

Background

The kidney is a major organ involved in calcium (Ca²⁺) metabolism. Ca²⁺ is transported through renal tubular epithelial cells. The intracellular free calcium concentration ([Ca²⁺]_i) is tightly controlled at a low concentration, but transient increases and oscillations in [Ca²⁺]_i are induced by various conditions. In this study, we investigated the mechanisms underlying the spontaneous [Ca²⁺]_i oscillations observed in MDCK cells.

Methods

[Ca²⁺]_i was monitored in fura-2-loaded Madin-Darby canine kidney (MDCK) cells using a calcium imaging system. We investigated the mechanism by which [Ca²⁺]_i changed by applying drugs or by changing the extracellular Ca²⁺ concentration.

Results

Spontaneous [Ca²⁺]_i oscillations occurred in MDCK cells. The oscillations occurred irregularly and were not transmitted to neighboring cells. Spontaneous [Ca²⁺]_i oscillations in MDCK cells were initiated by Ca²⁺ release from ryanodine/IP₃-sensitive intracellular calcium stores, and their frequency was largely unaffected by the extracellular Ca²⁺ concentration. Moreover, the frequency of the oscillations was increased by extracellular nucleotide, but was decreased when the nucleotides were removed.

Conclusions

Our study suggested that [Ca²⁺]_i release from ryanodine/IP₃-sensitive intracellular calcium stores mediates spontaneous [Ca²⁺]_i oscillations in MDCK cells. Calcium oscillations may be associated with the function of the renal tubular epithelial cells.     

Inositol (1,4,5)-trisphosphate dynamics and intracellular calcium oscillations in pancreatic beta-cells

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.     

Parathyroid Hormone (1-34) Receptor-Binding and Second-Messenger Response in Rat Incisor Odontoblasts

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca²⁺ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca²⁺ as a second messenger were evident. Received: 12 March 1997 / Accepted: 26 June 1997     

The effects of calcium antagonists and prostaglandin El on isolated canine coronary arterial tension

The effects of calcium antagonists (nifedipine, nicardipine, diltiazem, and verapamil) and prostaglandin E₁ (PGE₁) on the tension of isolated canine coronary arterial strips were studies. In a solution containing 20 mEq/L of K⁺, 127 mEq/L of Na⁺, the tension was increased by 500–1,000 mg with 4 mEq/L of Ca²⁺. This increase in tension was suppressed by Ca-antagonists and PGE₁ dose-dependently. Nifedipine 10^–5 M, nicardipine 3 X 10^–7 M, diltiazem 3 X 10^–6 M, and verapamil 3 X 10^–6 M completely suppressed the increased tension. The maximal suppression of the tension produced by PGE₁ was about 40% at 10^–10 M. In 20 mEq/L K⁺ solution (0 mEq/L Ca²⁺, 37°C), the reduction of the Na⁺ concentrations from 127 mEq/L to 12 mEq/L increased the tension by 50 to 100 mg. This increase in tension was not suppressed by Ca-antagonists or PGEI. In conclusion, this study demonstrated that Ca-antagonists and PGE₁ suppressed an increase in the tension caused by Ca²⁺ but did not suppress an increase in the tension caused by Na⁺ reduction.     

Intracellular free calcium mediates glioma cell detachment and cytotoxicity after photodynamic therapy

Photofrin photodynamic therapy (PDT) caused a dose-dependent decrease of enzymatic cell detachment by trypsin/ethylenediamine tetra-acetic acid (EDTA) in human glioma U251n and U87 cells. This happened coincidently with the increase of intracellular free calcium ([Ca²⁺]_i). Thapsigargin, which increased [Ca²⁺]_i, induced further decrease in enzymatic cell detachment and increased cytotoxicity. Opposite effects were observed when 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid tetrakis, an intracellular Ca²⁺ chelator, was used. PDT-induced changes in [Ca²⁺]_i and cell detachment were not blocked by calcium channel antagonists nickel (Ni²⁺) or nimodipine, nor were they altered when cells were irradiated in a buffer free from Ca²⁺ and magnesium (Mg²⁺), suggesting that [Ca²⁺]_i is derived from the internal calcium stores. Decreased cell migration was observed after PDT, as assessed by chemotactic and wound-healing assays. Our findings indicated that internal calcium store-derived [Ca²⁺]_i plays an important role in PDT-induced enzymatic cell detachment decrease and cytotoxicity. Cell migration may be affected by these changes.     

Propofol attenuates angiotensin II-induced vasoconstriction by inhibiting Ca2+-dependent and PKC-mediated Ca2+ sensitization mechanisms

Purpose

Angiotensin II (Ang II)-induced vascular contraction is mediated by Ca²⁺-dependent mechanisms and Ca²⁺ sensitization mechanisms. The phosphorylation of protein kinase C (PKC) regulates myofilament Ca²⁺ sensitivity. We have previously demonstrated that sevoflurane inhibits Ang II-induced vasoconstriction by inhibiting PKC phosphorylation, whereas isoflurane inhibits Ang II-induced vasoconstriction by decreasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) in vascular smooth muscle. Propofol also induces vasodilation; however, the effect of propofol on PKC-mediated myofilament Ca²⁺ sensitivity is poorly understood. The aim of this study is to determine the mechanisms by which propofol inhibits Ang II-induced vascular contraction in rat aortic smooth muscle.

Methods

An isometric force transducer was used to investigate the effect of propofol on vasoconstriction, a fluorometer was used to investigate the change in [Ca²⁺]_i, and Western blot testing was used to analyze Ang II-induced PKC phosphorylation.

Results

Ang II (10^?7?M) elicited a transient contraction of rat aortic smooth muscle, which was associated with an elevation of [Ca²⁺]_i. Propofol (10^?6?M) inhibited Ang II-induced vascular contraction (P?<?0.01) and increase in [Ca²⁺]_i (P?<?0.05) in rat aortic smooth muscle. Ang II also induced a rapid increase in [Ca²⁺]_i in cultured vascular smooth muscle cells, which was suppressed by propofol (P?<?0.05). Propofol (10^?6?M) attenuated Ang II-stimulated PKC phosphorylation (P?<?0.05).

Conclusion

These results suggest that the inhibitory effect of propofol on Ang II-induced vascular contraction is mediated by the attenuation of a Ca²⁺-dependent pathway and Ca²⁺ sensitivity through the PKC signaling pathway.