Electrophysiological differences between bone cell clones: membrane potential responses to parathyroid hormone and correlation with the cAMP response

Electrophysiological measurements on three clonally derived bone cell populations showed a positive correlation between longer-term hyperpolarizing membrane potential responses to parathyroid hormone (PTH) and an intracellular cAMP response to PTH. One clone (RCJ 1.20) had no sustained electrophysiological response and no cAMP response to PTH. Another clone (ROS 17/2.8) had both a sustained hyperpolarizing response and a cAMP response to PTH. The third clone (RCB 2.2) initially had both an electrophysiological response and a cAMP response to PTH, but both responses were lost after prolonged growth in culture. Application of dibutyryl cAMP to RCJ 1.20 and ROS 17/2.8 cells produced both transient and sustained hyperpolarizing responses. Application of isobutylmethylxanthine produced a sustained hyperpolarization. These results suggest that the hyperpolarizing response to PTH is related to a cAMP-mediated increase in Ca2+ conductance, which leads to an increase in Ca2+-activated K+ conductance. The pronounced membrane potential spikes and fluctuations that occur in some of the clonal lines were shown to be unrelated to the hyperpolarizing response to PTH. This was demonstrated by the lack of correlation between the occurrence of the spikes or fluctuations and the occurrence of the hyperpolarizing response to PTH in the various cell lines, by the lack of effect of PTH on the spikes and fluctuations, and by the lack of effect on the hyperpolarizing response to PTH of verapamil and quinine, both of which significantly reduce the spikes and fluctuations.     

Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells

Whole cell voltage clamp measurements using the patch technique on well-attached and well-spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak-induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum-free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.     

Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells.

Bovine parathyroid hormone (PTH), dibutyryl cAMP, and calcium each induce similar metabolic changes in isolated bone cells. PTH and calcium, but not dibutyryl cAMP, result in desensitization of osteoclastic and osteoblastic bone cells to PTH. In osteoblastic cells, calcium effects are specific for PTH receptor.adenylate cyclase complexes and responsiveness to other hormones is not reduced while in osteoclastic cells, small effects of high calcium on prostaglandin E1- and epinephrine-inducible cAMP accompany the large decreases seen in cAMP response to PTH. The membrane effects of calcium and of PTH appear to be independently regulated as PTH-induced desensitization can be initiated in the absence of calcium. In addition, calcium effects on PTH-sensitive adenylate cyclase follow a different calcium dose-response than PTH-like metabolic changes. These results suggest that the effect of calcium on the membrane is not directly related to its induction of PTH-like metabolic changes. A possible role of calcium as an in vivo regulator of bone cell sensitivity to PTH is discussed.     

Comparison of inhibition of bone resorption and escape with calcitonin and dibutyryl 3',5' cyclic adenosine monophosphate

Both parathyroid hormone (PTH) and calcitonin (CT) can increase the concentration of cyclic 3',5' adenosine monophosphate (cAMP) in fetal rat bone in organ culture. Moreover, dibutyryl cAMP (dbcAMP) can both stimulate and inhibit 45Ca release from such bones depending on dose and experimental conditions. In this study we compared dbcAMP and CT for their effects on bones pretreated with PTH. Both compounds produced transient inhibition of bone resorption followed by escape. Escape from dbcAMP was independent of prostaglandin synthesis, since it occurred both in the presence and absence of indomethacin, a prostaglandin cyclo-oxygenase inhibitor.     

Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis.

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.     

Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone

Measurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long-term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60-sec duration. In addition, increases in extracellular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long-term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+ equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+-dependent K+ channel, resulted in long-term depolarization of the mean membrane potential, and a long-term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+ entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+-dependent K+ membrane channels. Under an extracellular [Ca2+] of 1.8 mM, the application of prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2 did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2 effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30-sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long-term electrophysiological responses to hormone application, with only the long-term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long-term calcium transport responses to hormone application.     

A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP

It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast-like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two-receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts.     

Bone formation and calcification by isolated osteoblastlike cells

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.     

The effect of prostaglandin I2 and 6a-Carba-PGI2 on the motility of isolated osteoclasts

We have recently found that calcitonin (CT), a hormone which inhibits osteoclastic bone resorption, completely abolishes the normally intense cytoplasmic movement of isolated osteoclasts. We have also found that prostaglandin (PG)I2 causes an identical change in behaviour. In this paper we extend our investigations into the mode of action of PGI2 and a stable analogue, 6a-Carba-PGI2. We found that, unlike CT which causes prolonged immotility in osteoclasts, the effect of PGI2 and 6a-Carba-PGI2 were transient. Our results suggest that the transient nature of the inhibition was neither caused by inactivation of these compounds, nor was it due to production in the cultures of an osteoclastic stimulator. CT, PGI2 and 6a-Carba-PGI2 all appear to operate by increasing the intracellular cyclic AMP level. We found no refractoriness to either CT, dibutyryl cyclic AMP or 8-bromo cyclic AMP, and neither PGI2 nor 6a-Carba-PGI2 affected the sensitivity of osteoclasts to CT or dibutyryl cyclic AMP. This implies that refractoriness of osteoclasts to PGI2 and 6a-Carba-PGI2 develops at some stage in the interaction between PG and cell proximal to cyclic AMP production. We also found that there was cross-tachyphylaxis between PGI2 and 6a-Carba-PGI2, and this suggests that these two compounds share a receptor site on osteoclasts.     

氨基酸对蟾蜍卵母细胞膜电位的影响及其作用机制

本文采用微电极细胞内记录方法观察了23种氨基酸处理后蟾蜍卵母细胞膜电位的变化。丙氨酸、亮氨酸和赖氨酸可致膜电位去极化和膜K~+通透性降低,而色氨酸可致膜电位超极化和膜K~+通透性增加。其他19个氨基酸未见对膜电位有何作用。丙氨酸、亮氨酸和赖氨酸于引起去极化同时,还使卵母细胞孕酮含量成倍增加和卵母细胞趋于成熟。这些作用可被1mmol/L 丁酰 cAMP(db-cAMP)或 50μmol/L 雌二醇完全阻断。另一方面,色氨酸能阻断孕酮(10μmol/L)所致的去极化,但对 db-cAMP 所致的超极化无作用。本文讨论了氨基酸和甾类激素对卵母细胞成熟的调节作用。     

The effects of calcitonin, parathyroid hormone and prostaglandin E2 on cyclic AMP levels of isolated osteoclasts

Cyclic AMP (cAMP) levels were measured in both isolated and attached osteoclasts. The level of cAMP was 0.1 pmol/10(5) osteoclasts. No change in cAMP level of osteoclasts could be detected following calcitonin treatment. Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) treatment stimulated cAMP production in proportion to alkaline phosphatase levels and divergent to acid phosphatase levels. This indicates that osteoblasts, not osteoclasts, were responsive to PTH and PGE2.     

The effect of prostaglandin I2 and 6a-Carba-PGI2 on the motility of isolated osteoclasts

We have recently found that calcitonin (CT), a hormone which inhibits osteoclastic bone resorption, completely abolishes the normally intense cytoplasmic movement of isolated osteoclasts. We have also found that prostaglandin (PG)I₂ causes an identical change in behaviour. In this paper we extend our investigations into the mode of action of PGI₂ and a stable analogue, 6a-Carba-PGI₂. We found that, unlike CT which causes prolonged immotility in osteoclasts, the effect of PGI₂ and 6a-Carba-PGI₂ were transient. Our results suggest that the transient nature of the inhibition was neither caused by inactivation of these compounds, nor was it due to production in the cultures of an osteoclastic stimulator. CT, PGI₂ and 6a-Carba-PGI₂ all appear to operate by increasing the intracellular cyclic AMP level. We found no refractoriness to either CT, dibutyryl cyclic AMP or 8-bromo cyclic AMP, and neither PGI₂ nor 6a-Carba-PGI₂ affected the sensitivity of osteoclasts to CT or dibutyryl cyclic AMP. This implies that refractoriness of osteoclasts to PGI₂ and 6a-Carba-PGI₂ develops at some stage in the interaction between PG and cell proximal to cyclic AMP production. We also found that there was cross-tachyphylaxis between PGI₂ and 6a-Carba-PGI₂, and this suggests that these two compounds share a receptor site on osteoclasts.     

Dopamine as a modulator of ionic transport and glycolytic fluxes in the gills of the chinese crab,Eriocheir sinensis

1. Dopamine and dibutyryl cAMP induce a significant hyperpolarization when the so-called anterior and posterior gills isolated from the crab Eriocheir sinensis acclimated to freshwater are perfused with the same saline on both sides.2. When compared to the anterior ones, the posterior gills show higher concentrations of fructose 2,6-biphosphate (Fru 2,6-bP) and a lower ATP/ADP ratio.3. Perfusion with a freshwater or seawater saline decreases both the level of Fru 2,6-bP and the ATP/JADP ratio whereas dopamine and dibutyryl cAMP significantly increase the Fru 2,6-bP content for the posterior and the anterior gills.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

TheBulla ocular circadian pacemaker

In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker of Bulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that: 1. Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization. 2. Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180 degrees in circadian time to depolarization. 3. Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection. 4. The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts. 5. The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3 X 10(-7) M inhibits light-induced phase shifts without blocking the photic response of the BRNs. The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.