Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells

The availability of Ca2+ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular [Ca2+] ([Ca2+]o) and modify their function. The effects of changing [Ca2+]o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca2+]o in a dose-dependent and reversible manner (ID50, approximately 2 mM Ca2+). RNA levels for aggrecan and type II collagen decreased with increasing [Ca2+]o (ID50, approximately 2.0 and 4.1 mM Ca2+, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca2+]o with ID50 values of approximately 2.9 and 1.6 mM Ca2+, respectively. These responses were rapid, in that increasing [Ca2+]o from 1.0 to more than 6 mM suppressed aggrecan RNA levels by about 50%, and lowering [Ca2+]o from 2.9 to 1.0 mM increased aggrecan RNA levels by about 300% within 4 h. As Ca2+ receptors (CaRs) mediate extracellular Ca2+ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.     

Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts

The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca(2+)(o), a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca(2+)(o) did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca(2+)(o) did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca(2+)(o)-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca(2+)(o) is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca(2+)(o) stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.     

Extracellular Ca(2+)-sensing receptors modulate matrix production and mineralization in chondrogenic RCJ3.1C5.18 cells

Previous studies in chondrogenic RCJ3.1C5.18 (C5.18) cells showed that growth of these cells at high extracellular Ca(2+) concentrations ([Ca(2+)](o)) reduced the expression of markers of early chondrocyte differentiation. These studies addressed whether raising [Ca(2+)](o) accelerates C5.18 cell differentiation and whether Ca(2+) receptors (CaRs) are involved in coupling changes in [Ca(2+)](o) to cellular responses. We found that high [Ca(2+)](o) increased expression of osteopontin (OP), osteonectin, and osteocalcin, all markers of terminal differentiation, in C5.18 cells and increased the production of matrix mineral. Overexpression of wild-type CaR cDNA in C5.18 cells suppressed proteoglycan synthesis and aggrecan RNA, two early differentiation markers, and increased OP expression. The sensitivity of these parameters to changes in [Ca(2+)](o) was significantly increased, as indicated by left-shifted dose-responses. In contrast, stable expression of a signaling-defective CaR mutant (Phe707Trp CaR) in C5.18 cells, presumably through dominant-negative inhibition of endogenous CaRs, blocked the suppression of aggrecan RNA levels and proteoglycan accumulation and the enhancement of OP expression by high [Ca(2+)](o). These data support a role for CaRs in mediating high [Ca(2+)](o)-induced differentiation of C5.18 cells.     

Constitutive activity of the osteoblast Ca2+-sensing receptor promotes loss of cancellous bone

Changes in extracellular [Ca2+] modulate the function of bone cells in vitro via the extracellular Ca2+-sensing receptor (CaR). Within bone microenvironments, resorption increases extracellular [Ca2+] locally. To determine whether enhanced CaR signaling could modulate remodeling and thereby bone mass in vivo, we generated transgenic mice with a constitutively active mutant CaR (Act-CaR) targeted to their mature osteoblasts by the 3.5 kb osteocalcin promoter. Longitudinal microcomputed tomography of cancellous bone revealed reduced bone volume and density, accompanied by a diminished trabecular network, in the Act-CaR mice. The bone loss was secondary to an increased number and activity of osteoclasts, demonstrated by histomorphometry of secondary spongiosa. Histomorphometry, conversely, indicates that bone formation rates were unchanged in the transgenic mice. Constitutive signaling of the CaR in mature osteoblasts resulted in increased expression of RANK-L (receptor activator of nuclear factor-kappaB ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR.     

Activation of dihydropyridine-sensitive calcium channels and biphasic cytosolic calcium responses by angiotensin II in rat adrenal glomerulosa cells

The steroidogenic actions of angiotensin II (AII) and increased extracellular K+ concentrations [( K+]) in rat adrenal glomerulosa cells are selectively enhanced by the voltage-sensitive calcium channel agonist Bay K 8644 (BK 8644). The relationship between these effects of the dihydropyridine agonist and cytosolic calcium concentration [( Ca2+]i) was investigated in rat and bovine glomerulosa cells. In the rat glomerulosa cells, AII and increased [K+] elicited rapid elevations of [Ca2+]i with distinctive temporal characteristics. Whereas the [Ca2+]i response to [K+] declined to basal over 2-3 min, addition of 10 nM AII caused a biphasic increase in [Ca2+]i, with a rapid transient rise followed by a lower plateau phase that remained above basal for several minutes. BK 8644 alone did not affect [Ca2+]i, but at low concentrations (30 nM) increased the magnitude and duration of the [Ca2+]i response elicited by progressive elevation of extracellular [K+] to 12 mM. In AII-stimulated glomerulosa cells, 30 nM BK 8644 enhanced both phases of the cytosolic calcium response, with a more marked effect on the sustained plateau phase. In contrast to its prominent actions in rat glomerulosa cells, BK 8644 had no effect on AII-stimulated rises in [Ca2+]i in bovine glomerulosa cells, and only slightly enhanced their minor [Ca2+]i responses to potassium. These studies provide evidence that AII activates dihydropyridine-sensitive voltage-sensitive calcium channels in rat, but not bovine, adrenal glomerulosa cells. They also suggest that enhancement by BK 8644 of agonist-stimulated [Ca2+]i changes is responsible for its synergistic effects on aldosterone responses to potassium and AII in rat glomerulosa cells and emphasize the importance of the sustained phase of the cytosolic calcium signal in the steroidogenic action of AII.     

Parathyroid hormone (PTH)-induced intracellular Ca2+ signalling in naive and PTH-desensitized osteoblast-like cells (ROS 17/2.8): pharmacological characterization and evidence for synchronous oscillation of intracellular Ca2+

We showed recently that the initial peak cytosolic ionized calcium ([Ca2+]i) response to PTH (2-min exposure) is preserved relative to the cAMP response in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) desensitized by 72-h exposure to PTH. We attempted in the present studies to determine the mechanisms for preservation of the [Ca2+]i response and to explore the effects of longer PTH rechallenges. The [Ca2+]i response to a 20-min perifusion with rat PTH [rPTH-(1-34)] was monitored by aequorin luminescence in both naive and PTH-desensitized ROS 17/2.8 cells. The responses of both naive and desensitized cells consisted of two phases: an initial peak, followed by an intermediate plateau that was sustained in the presence of PTH. We observed in the naive cell populations synchronous oscillations in [Ca2+]i concentration during this second phase (amplitude, 10-60 nM; frequency, 1-3/100 sec). These oscillations were maintained through extracellular calcium (EC Ca2+) entry; the initial peak was the result of Ca2+ release from intracellular stores. In desensitized cells, these two phases could not be clearly separated with respect to Ca2+ source, but, as we showed before, exhibited an enhanced dependence on EC Ca2+ entry for the response to PTH. Nevertheless, in the desensitized cells, the sustained [Ca2+]i response was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]i rise in both naive and desensitized cells. Protein kinase-C activity did not appear to be required for either phase of the PTH-induced [Ca2+]i response. Exposure to cholera toxin attenuated the [Ca2+]i response to hormone in both naive and desensitized cells, more markedly in the latter. Cholera toxin treatment dramatically increased basal cAMP levels in both cell preparations; PTH-stimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]i rise in desensitized cells results primarily from the diminished regulation of EC Ca2+ entry by the cAMP response limb. The attenuated sustained oscillatory behavior observed in desensitized cells upon rechallenge with hormone may be the result of reduced phosphoinositide turnover and reduced Ca2+-stimulated Ca2+ release. Thus, the [Ca2+]i response to PTH in osteoblast-like cells is complex and modulable and seems to provide a number of ways to regulate intracellular metabolism under various conditions. We speculate that this plasticity of the [Ca2+]i response to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage.     

Desensitization of rat osteoblast-like cells (ROS 17/2.8) to parathyroid hormone uncouples the adenosine 3',5'-monophosphate and cytosolic ionized calcium response limbs.

We have investigated the effects of PTH-induced desensitization on second messenger interactions in the rat osteosarcoma cell line ROS 17/2.8. Adenylate cyclase activation was assessed by accumulation of immunoassayable cAMP, and cytosolic calcium ion ([Ca2+]i) concentrations were measured in adherent perifused cells loaded with the Ca2(+)-sensitive bioluminescent protein aequorin. Preexposure to rat PTH-(1-34) [rPTH-(1-34); 10(-8) M for 48 h, then 10(-7) M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10(-9)-10(-7) M rPTH-(1-34)], but the peak PTH-induced rise of [Ca2+]i was not diminished significantly (0-20%). Nevertheless, we did observe other changes in the PTH-induced [Ca2+]i response. Exposure of treated cells to (Bu)2cAMP nearly abolished the [Ca2+]i response to PTH (greater than 80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (less than 35% reduction). Treated cells also had a blunted [Ca2+]i response to PTH in the presence of low extracellular calcium (greater than 60% reduction), but in the naive cells, low extracellular Ca2+ did not significantly diminish the peak PTH-induced [Ca2+]i rise, although low extracellular Ca2+ dramatically reduced the area under this [Ca2+]i transient (greater than 50%). Low extracellular Ca2+ had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2 alpha. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-maximum [Ca2+]i concentration was significantly increased (greater than 100%), and the area under the [Ca2+]i transient was diminished. These alterations in the [Ca2+]i response of treated cells were not observed upon challenge with bradykinin or prostaglandin F2 alpha. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(1-34) are not obligatorily coupled; 2) the response of naive cells to PTH includes both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+; and 3) pretreatment of these cells with rPTH-(1-34) augments the dependence on Ca2+ entry during hormone rechallenge. We propose that the preserved PTH-stimulated [Ca2+]i rise in treated cells results partly from loss of cAMP-mediated inhibition of extracellular Ca2+ entry.     

Intracellular free calcium in response to oxytocin in pig endometrial cells

Intracellular free calcium concentration ([Ca2+]i) in response to oxytocin (OT) was studied in stromal, glandular epithelial and luminal epithelial cells obtained from the endometrium of gilts 16 days post-estrus. The amplitude of increased [Ca2+]i in response to 100 nM OT was greatest in stromal cells, intermediate in glandular epithelial cells and not evident in luminal epithelial cells. During continuous OT administration, stromal cells responded initially with a synchronous spike of [Ca2+]i that did not require extracellular Ca2+ and then displayed spontaneous asynchronous [Ca2+]i spikes that required extracellular Ca2+. Each cell possessed its own characteristic response. Increasing concentrations of OT induced an increasing percentage of stromal cells responding, with some cells having nearly equal [Ca2+]i responses at all concentrations and others having graded [Ca2+]i responses as the concentration of OT increased. These results are consistent with the proposed mechanism of OT action in pig endometrium involving activation of phosphoinositide-Ca2+ signaling pathway.     

Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells.

The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.     

Stimulation of muscarinic receptors raises free intracellular Ca2+ concentration in rat ventricular myocytes

The effect of carbachol on free intracellular calcium concentration, ([Ca2+]i) and on intracellular hydrogen concentration (pHi) was determined from fluorescence signals obtained from rat ventricular myocytes. Application of carbachol (300 mumol/l) to quin2-loaded myocytes bathed in 2 mmol/l Ca2+-containing solution caused [Ca2+]i to increase within 7-10 minutes from 182 +/- 9 to 212 +/- 11 nmol/l (n = 4). Carbachol acted via stimulation of muscarinic receptors because atropine (1 mumol/l) either prevented or abolished the increase in [Ca2+]i. Carbachol also produced a positive inotropic effect in rat papillary muscles contracting isometrically at a frequency of 0.5 Hz and enhanced contracture in resting preparations in the presence of high extracellular Ca2+ concentration ([Ca2+]o) (20 mmol/l). The effect of carbachol on [Ca2+]i was dependent on [Ca2+]o. In the presence of 10 mmol/l [Ca2+]o, the increase in [Ca2+]i was about two times that elicited by carbachol when bath [Ca2+]o was 2 mmol/l. Reduction of [Ca2+]o to 50 mumol/l abolished the carbachol effect but did not prevent caffeine-induced Ca2+ release. The carbachol-induced rise in [Ca2+]i remained unchanged in the presence of either 10 mmol/l caffeine or 1 mumol/l ryanodine. In the absence of extracellular Na+ concentration [( Na+]o), carbachol no longer produced an increase in [Ca2+]i of cardiomyocytes and failed to enhance Na+-withdrawal contracture of the rat papillary muscle. In contrast to the effect on [Ca2+]i, carbachol did not produce any change in pHi as determined from fluorescence signals obtained from rat ventricular myocytes loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of calcitonin on 3',5'-cyclic adenosine monophosphate and calcium second messenger generation and osteoblast function in UMR 106-06 osteoblast-like cells.

The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Distinct intracellular Ca2+ response to extracellular adenosine triphosphate in pancreatic beta-cells in rats and mice

Extracellular adenosine triphosphate (ATP) has distinct effects on insulin secretion from pancreatic β-cells between rats and mice. Using a confocal microscope, we compared changes between rats and mice in cytosolic free calcium concentration ([Ca²⁺]_c) in pancreatic β-cells stimulated by extracellular ATP. Extracellular ATP (50 μM) induced calcium release from intracellular calcium stores by activating P2Y receptors in both rat and mouse β-cells. The intracellular calcium release stimulated by extracellular ATP is significantly smaller in amplitude and longer in duration in rat β-cells than in mouse. In response to extracellular ATP, rat β-cells activate store-operated calcium entry following intracellular calcium release. This response is lacking in mouse β-cells. Rat and mouse β-cells both responded to 9 mM glucose by increasing [Ca²⁺]_c. This increase, however, was pronounced only in the rat β-cells. In 9 mM glucose, extracellular ATP induced a pro-nounced calcium release above the increased level of [Ca²⁺]_c in rat β-cells. In mouse β-cells, however, extracellular ATP did not exhibit calcium release on top of the increased level of [Ca²⁺]_c in 9 mM glucose. These results demonstrate distinct responses between rat and mouse β-cells to extracellular ATP under the condition of low and high glucose. Considering that extracellular ATP inhibits insulin secretion from mouse β-cells but stimulates insulin secretion from rat β-cells, we suggest that store-operated Ca²⁺ entry may be related to exocytosis in pancreatic rat β-cells.     

Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions.

Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca2+, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca2+ concentration, [Ca2+]i, is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP3), a second messenger that can evoke Ca2+ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca2+ and InsP3 in freshly isolated pairs or small clusters of rat hepatocytes. The Ca2+ indicator fura-2 was used to monitor transjunctional diffusion of Ca2+ directly or to detect passage of InsP3 by localized Ca2+ release. Fura-2 injected as the free acid passed between cells. Injection of InsP3 or CaCl2 immediately increased [Ca2+]i in the injected cell (peak values less than 1 microM), and [Ca2+]i increased rapidly in contacting cells (within seconds). The initial rise in [Ca2+]i induced by InsP3 was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca2+. In the coupled cells the regions of greatest increase were not necessarily near the contact zone. In contrast, the rise induced in [Ca2+]i by CaCl2 injection when cells were bathed in normal Ca2+ was always more diffuse than with InsP3 injection, and in cells coupled to a cell injected with CaCl2 the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP3 (or an active metabolite, but not Ca2+) diffused between cells to cause localized release of Ca2+ from intracellular stores. Ca2+ injection induced a rise in [Ca2+]i in coupled cells even when cells were maintained in Ca2+-free saline, suggesting that changes in [Ca2+]i seen in adjacent cells were due to transjunctional diffusion from the injected cell and not to uptake from the extracellular solution. However, in Ca2+-free saline, [Ca2+]i distribution was nonuniform, indicating that Ca2+-releasing mechanisms contribute to the observed changes. No increase in [Ca2+]i was seen in adjacent cells when Ca2+ was injected after treatment with the uncoupling agent octanol (500 microM), which itself did not change [Ca2+]i. These data provide evidence that the second messengers Ca2+ and InsP3 can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.     

Alpha-adrenergic modification of the Ca2+ transient and contraction in single rat cardiomyocytes.

Intracellular Ca2+ transients and contraction were measured simultaneously in single rat cardiomyocytes loaded with the fluorescent Ca2+ indicator fura-2, using a recently described high-speed digital imaging method (O'Rourke et al., 1990, Am J Physiol 259: H230-H242). In cardiomyocytes electrically-stimulated at 1 Hertz, alpha-adrenoceptor activation in the presence of beta-adrenoceptor blockade resulted in enhanced cell shortening associated with an increase in the amplitude of the cytosolic Ca2+ transient. Both effects developed in parallel over a 10-min time period and occurred without a change in the half-times for decay of Ca2+ or relaxation of the cell. To determine if the increase in contractility was proportional to the increase in peak cytosolic Ca2+, the effect of raising extracellular Ca2+ ([Ca2+]o) from 0.5 to 3 mM was examined in the absence and presence of alpha-adrenoceptor activation. At [Ca2+]o concentrations up to 1 mM, alpha-adrenoceptor-mediated effects on contraction were directly correlated with changes in peak cytosolic Ca2+ and resembled the effect of raising [Ca2+]o alone. In 2 and 3 mM [Ca2+]o, peak cytosolic Ca2+ approached a maximal level and alpha-adrenoceptor activation induced a slight enhancement in the extent of shortening in the absence of a detectable alteration of the Ca2+ transient. In contrast, under similar conditions, beta-adrenergic effects on shortening never exceeded those of alpha-adrenoceptor activation, although much higher peak cytosolic Ca2+ concentrations were achieved at high [Ca2+]o. The results suggest that the mechanism underlying the positive inotropic effect of alpha-adrenergic stimulation in rat ventricular cells is primarily dependent on an enhancement of the cytosolic Ca2+ transient, although there is also an increase in the myofibrillar response to intracellular Ca2+ under the condition of high extracellular Ca2+.     

Functional and structural impairment in human, rat and guinea-pig atrial muscle in response to in vitro calcium overload: a cytochemical study on cellular calcium distribution.

Critical accumulation of cellular calcium in ischaemic myocardium is involved in irreversible cell damage. In human right atrial trabeculae and in rat and guinea-pig left atria, we investigated whether direct calcium overload by increasing the extracellular calcium concentration, [Ca2+]o, leads to similar impairment of function and ultrastructure as observed after ischaemia. The force of contraction was measured during two consecutive cumulative increases in [Ca2+]o (1-25 mmol/l) separated by 30 min of incubation at low [Ca2+]o. Compared to the first Ca2+ challenge, the positive inotropic effect of increasing [Ca2+]o was depressed during the second one and the after-contractions, and the increase in resting tension developing with high [Ca2+]o tended to be larger. The ultrastructure of the tissue fixed immediately after excision was well preserved. When fixed after the second Ca2+ challenge, half of the cells were severely damaged with various signs of cellular Ca2+ overload similar to those observed after ischaemic damage: the sarcolemma lost its Ca(2+)-binding properties, sarcomeres showed contraction band necrosis, the mitochondria had disrupted cristae and contained either large clusters of Ca2+ precipitate or amorphous densities (Jennings granules). In many cells, calcium precipitates were present in the cytoplasm. The morphological and functional changes were similar in the three species studied. Our results suggest that the deterioration of atrial myocardium after challenge with high [Ca2+]o or after severe ischaemia may be traced back to a common mechanism, i.e. the sarcolemma loses its competence as a permeable barrier for Ca2+ and therefore facilitates excessive Ca2+ entry. However, for the direct demonstration of calcium precipitates as a sign of cytosolic Ca2+ overload, high [Ca2+]o are required with are not normally present in the myocardium.     

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca2+ concentration ([Ca2+]i) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH greater than 10 pM elicited increases in [Ca2+]i in identified cells but not in others. In contrast, depolarization induced by 50 mM K+ increased [Ca2+]i in all cells. Ca2+ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca2+]i fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca2+ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca2+]i. Removal of extracellular Ca2+ or addition of the dihydropyridine Ca2+-channel blocker nitrendipine completely blocked the secondary rise in [Ca2+]i but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K+-induced increases in [Ca2+]i in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca2+ influx was permitted either by allowing a secondary rise to occur or by producing a Ca2+ transient by depolarizing the cells with 50 mM K+. It therefore appears that the response to GnRH consists of an initial phase of Ca2+ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca2+ influx through nitrendipine-sensitive Ca2+ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.     

Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs.

The relationships between the activation status of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perfused rat gonadotrophs during stimulation by high extracellular K+ concentration ([K+]e) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [K+]e to 50 mM evokes an on-off secretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+]e is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+]e and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch clamp experiments. In addition, the profile of the LH secretory response is similar to that of the response of intracellular Ca2+ concentration ([Ca2+]i) in K(+)-stimulated cells. In Ca2(+)-deficient medium, the effect of high [K+]e is abolished; subsequent elevation of [Ca2+]e during the K+ pulse is followed by restoration of the on response, but with reduced magnitude. Agonist stimulation during the steady-state phase of the [K+]e pulse or after repetitive stimulation by high [K+]e elicited biphasic [Ca2+]i and secretory responses with a significantly reduced plateau phase; conversely, K(+)-induced LH release was reduced in cells treated with desensitizing doses of GnRH. These findings indicate that depolarization-induced changes in the status of voltage-sensitive Ca2+ channels determine the profiles of [Ca2+]i and LH responses to stimulation by high [K+]e; the initial activation of dihydropyridine-sensitive Ca2+ channels is clearly dependent on membrane potential, whereas their subsequent inactivation depends on increased [Ca2+]i. Such inactivation of voltage-sensitive Ca2+ channels also occurs during GnRH action and may represent an additional regulatory mechanism to limit the entry of extracellular Ca2+ during prolonged or frequent agonist stimulation.