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Endocrinology Vol. 139, No. 8 3492-3502
Copyright © 1998 by The Endocrine Society

ARTICLES

Calcitonin Gene-Related Peptide Stimulates Potassium Efflux through Adenosine Triphosphate-Sensitive Potassium Channels and Produces Membrane Hyperpolarization in Osteoblastic UMR106 Cells1

Tomoyuki Kawase and Douglas M. Burns

Department of Pharmacology (T.K.), Niigata University School of Dentistry, Niigata 951, Japan; and Medical Research Service (D.M.B.), Kansas City Department of Veterans Affairs Medical Center and Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Missouri 64128

Address all correspondence and requests for reprints to: Tomoyuki Kawase, D.D.S., Ph.D., Department of Pharmacology, Niigata University School of Dentistry, 2–5274 Gakkicho-dori, Niigata-city, Japan 951. E-mail: kawase@dent.niigata-u.ac.jp or dburns{at}kuhub.cc.ukans.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits 45Ca2+ uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (KATP) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01–100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a KATP channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective KATP channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a KCa channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01–100 nM) decreased intracellular K+ concentrations ([K+]i) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of KATP channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT) gene-related peptide (CGRP), an amidated 37-residue neuropeptide produced from the CT gene (1, 2), is prominent in sensory nerve fibers within the bone, marrow, and periosteal regions of skeletal tissue (3, 4, 5, 6, 7, 8, 9, 10) and may play an important regulatory role in bone metabolism. Several studies have demonstrated the efferent release of CGRP and other neuropeptides from capsaicin-sensitive peptidergic fibers (11, 12, 13, 14, 15) in several tissues, and indirect evidence has suggested that this also occurs in bone and its adjacent tissues (3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). A recent report from Imai et al. (19) has provided striking ultramorphological support for this idea by demonstrating in rat that osseous CGRP-containing axons possess terminal structures that directly contact metaphyseal osteoblasts and osteoclasts. Furthermore, this study demonstrated that these CGRP-containing peptidergic fibers reinnervate bony surface at the same time that osteoblasts begin to be recruited and activated during osteogenic repair of experimentally eroded bone (19).

Tippins et al. (20) and Roos et al. (21) originally reported that pharmacological doses of CGRP inhibit osteoclastic bone resorption, but several studies (22, 23, 24) now indicate that this effect is mediated by cross-binding to CT receptors (with a 300-fold lower biopotency). Michelangeli et al. (25) first convincingly demonstrated that exogenous CGRP stimulates the production of cAMP in osteogenic osteosarcoma cells and further demonstrated that the ability of cellular fractions, enzymatically released from bone, to respond to CGRP correlates directly with the degree to which they possess the osteoblastic phenotype (26). Numerous additional studies have now verified that low doses of CGRP are indeed able to produce modest (2- to 3-fold or higher) increases in cAMP in both osteosarcoma and primary osteoblastic cells (7, 27, 28, 29, 30). The microhistologic and ultramorphological data and the overall pattern of in vitro CGRP action on skeletal cells has led to the proposal that this neuropeptide exerts anabolic actions on skeletal and mineral metabolism, largely by recruiting and/or activating osteoblastic cells (7, 16, 17, 18, 28, 29, 31). The response of cultured osteoblastic cells to low levels of CGRP seems to be mediated by a specific CGRP receptor on osteoblasts (32) (also Burns, unpublished observations). CGRP has previously been shown to stimulate the formation of osteogenic cell colonies from cultured rat bone marrow stromal cells (17) and, more recently, to stimulate in vitro mineralization in osteoblastic cell cultures (31). Furthermore, CGRP is associated in rats with heterotropic bone formation induced by demineralized bone matrix (18), with the calcification of secondary and tertiary dentin (3, 16), with repair of damaged metaphyseal bone in an adjuvant arthritic rat model (19), and with repair of tibial fractures (33). Most recently, the reports from Vignery and co-workers have demonstrated additional bioactivity for CGRP on osteoblasts, and their pilot experiments strongly suggest that exogenous CGRP produces appreciable stimulation of bone formation in rat, at least in part, through direct stimulation of osteoblastic parameters (30, 34, 35).

It is now important to understand how CGRP exerts its neurogenic actions on osteoblasts. An analogous situation is found in vascular smooth muscle, where CGRP acts as a rapid and potent vasodilator. Originally, this vascular action of CGRP was explained solely by cAMP-dependent mechanism(s) (36, 37, 38); however, a more recent mechanism has been demonstrated whereby CGRP directly activates ATP-sensitive potassium (KATP) channels to induce hyperpolarization of the plasma-membrane and then produces relaxation of the smooth muscle cell (39, 40). In osteoblastic cells, the existence of calcium-activated potassium (KCa) channels has been demonstrated by electrophysiological studies (41, 42), and KATP channels have now been identified to be present and capable of playing a role in osteoblastic physiology (43, 44). It has become important to develop an understanding of the properties of osteoblastic KATP channels and the mechanism(s) that regulate(s) related cellular parameters, such as membrane potential (Em).

In previous studies (45, 46), we interpreted data from pharmacological experiments with cultured osteoblastic cells as strongly suggesting that osteoblasts possess both L-type Ca2+ and KATP channels and that CGRP acts on one or both of these channels to attenuate Ca2+ influx across the plasma membrane. To further investigate the mechanism mediating CGRP’s actions in osteoblastic cells, we now directly examine CGRP’s ability to influence cellular Em and to alter intracellular K+ concentration ([K+]i) in UMR106 osteoblast-like cells, by using fluorescent dye assays. Our results indicate that CGRP primarily acts on KATP channels to first induce a significant K+ efflux that, in turn, produces hyperpolarization of the plasma membrane. These data strongly support our previous proposal that these actions of CGRP cause the observed attenuation of transmembrane Ca2+ influx.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat CGRP (Peptide Institute, Osaka, Japan) was dissolved in distilled water (DW) to a concentration of 100 µM. Glibenclamide (Glib) and tetraethylammonium (TEA) chloride were obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions of pinacidil (Pina; Eli Lilly Co., Indianapolis, 2IN) and Glib were prepared in dimethylsulfoxide (DMSO) to 20 mM and 2 mM, respectively. Bis-(1, 3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] (Molecular Probes, Inc., Eugene, OR) was dissolved in ethanol (EtOH) to 20 mM stock, while the acetoxymethyl ester of PBFI (PBFI-AM) (Molecular Probes, Inc.) was dissolved in DMSO to 1 mM stock. Valinomycin (Calbiochem-Novabiochem International, San Diego, CA) and nigericin (Sigma) were dissolved in EtOH to 1 mM stock and EtOH:DMSO (1:1) to 5 mM stock, respectively. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89–2HCl), from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA), was dissolved in DMSO to a stock concentration of 5 mM. Unless otherwise specified, all stock solutions were diluted in physiological saline solution (PSS) or appropriate aqueous solution, to the required working concentration, immediately before use.

Cells and cell culture
Rat osteosarcoma UMR106 cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan) and maintained in DMEM (GIBCO, Grand Island, NY) supplemented with 10% FCS (FCS; Filtron, Brooklyn, Australia) in humidified 5% CO2-95% air at 37 C. For all experiments reported here, cells were seeded at a density of 3 x 105 cells/4 ml·60-mm dish and cultured for 2 days in DMEM+10% FCS and for 1 additional day in DMEM containing 2% FCS. For Em measurements, cells were seeded on glass coverslips placed on the bottoms of 60-mm dishes.

Measurements of Em
The methodology introduced by Civitelli et al. (47) was slightly modified and applied here. After rinsing with warmed PSS, cells attached on coverslips were preincubated in PSS containing 0.5 µM DiBAC4(3) and 0.1% FCS for 40–50 min and then directly subjected to the fluorometric assay using a Shimazu RF-300 spectrofluorometer (Kyoto, Japan) equipped with a thermostatically controlled (37 C) cuvette chamber and an electric stirrer. Excitation wavelength was 493 nm (ND filter no. 4) and emission wavelength 513 nm (ND filter no. 16), with 10 nm bandwidth for both. At these wavelengths, the signal was least influenced by coverslips. For fluorescence intensity (FI; y-axis), the same scale (arbitrary unit) was used throughout all the figures presented.

We attempted, but could not achieve, calibration between changes in the FI emission of DiBAC4(3) and calculated mV of the cellular Em. Unfortunately, this bis-oxonol dye binds several ionophores (e.g. valinomycin or gramicidin) to form a fluorescent precipitate that severely interferes with attempts at calibrating the cellular FI signal (47) (also Kawase et al., unpublished observations).

Measurement of [K+]i
After rinsing with warmed PSS, cells were loaded with 1 µM PBFI-AM in PSS for 90 min in a CO2 incubator. Cells were then detached with 0.05% trypsin (GIBCO) plus 0.52 mM EDTA in Ca2+, Mg2+-free Dulbecco’s modified phosphate buffer saline [PBS(-)] and suspended in PSS at a density of 5–6 x 105 cells/ml. Cell fluorescence was measured by a dual-wavelength ratiometric method, essentially as described previously (48, 49): For excitation, both 338 and 380 nm (as isobestic point) were employed; and for emission, 465 nm was used for both excitation wavelengths. Bandwidths were fixed at 10 nm for both wavelengths.

Calibration of the PBFI signal
According to the method of Kasner et al. (48, 49), the combination of valinomycin (final concentration = 1 µM) and nigericin (final concentration = 5 µM) was added to cell suspensions to equally distribute K+, both inside and outside the plasma membrane, and to equilibrate the transmembrane pH gradient. Cells loaded with PBFI were incubated for 15 min in calibrating solution using different known [K+]o solution, and the FI ratio was measured. The resulting standard curve (not shown) was employed to calibrate the recorded PBFI signal vs. a [K+] standard curve.

2-D analysis of cell volume
Cells in subconfluent cultures were washed twice with warmed PSS and preincubated with PSS in the presence or absence of Glib for 10 min. Cells were then treated with CGRP in the presence or absence of Glib for 10 min on a warmed pad (approximately 37 C) and observed under an inverted microscope (Nikon, Tokyo, Japan). At the indicated treatment times, cells were photographed and subjected to 2-D analysis using NIH image software (version 1.55, NIH, Bethesda, MD) installed on a Macintosh computer.

Statistical analysis
Most results in figures are expressed as the mean ± SD of 5–15 independent samples, unless otherwise specified, and the statistical significance was calculated with two-tailed, unpaired Student’s t test. Each trace is representative of the data from those independent experiments. Only the data shown in Fig. 10Go were analyzed with paired Student’s t test (N = 20 cells in each experiment, and nearly identical results were obtained from three additional independent experiments).



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  		Figure 10. Photos, The effects of CGRP on the morphology of adherent subconfluent cultures of UMR106 cells. Cells were treated for 10 min, either with vehicle alone (A and B) or with CGRP (100 nM; C and D). A third (parallel) set of cells was pretreated with Glib (1 µM) for 10 min and then cotreated with Glib and CGRP for 10 min (100 nM; E and F). Examination of the photomicrographs reveals that CGRP produces noticeable retraction of the cellular cytoplasm leaving behind psuedopodia-like processes; and in many cells, the area of cellular cross-section noticeably decreases. Graph, The time-course for changes in cellular area in attached UMR106 cells. Cellular cross-sectional area was quantified by 2-D computer analysis, as described in Materials and Methods and plotted vs. treatment time. Note: After 10 min of treatment, the area of CGRP-treated cells (open squares) was significantly smaller (a) (P < 0.01 from a paired Student’s t test, N = 20) by approximately 17% than that of the vehicle control treatment (open circles; D vs. B) or than that of cells cotreated with Glib + CGRP (closed squares; D vs. F).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go demonstrates a dose-dependent effect of CGRP (0.01–100 nM) on the resting Em of DiBAC4(3)-loaded UMR106 cells attached to coverslips. The indicated concentrations of CGRP were applied in succession to the same population of cells, and the FI of DiBAC4(3) was recorded as a direct report of cellular Em. No less than 2 min elapsed between the observed end of one response (establishment of a new baseline) and the start of the next application. In this manner, Em changes could be traced cumulatively with direct reference to the original starting Em, as shown in Fig. 1AGo. CGRP elicited a discernible response within approximately 20 sec of application; however, the length of time required for full hyperpolarization to develop varied with the concentrations of CGRP applied. With 0.01 nM CGRP, full hyperpolarization was obtained within 2 min, but higher concentrations required longer for full expression. Continuous measurements with DiBAC4 demonstrate a progressive decline in the FI baseline that probably represents time-dependent photo-bleaching of the fluorescent dye itself; thus, there is a limit to how long an experiment can provide reliable data.



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  		Figure 1. A, The effects of CGRP on the plasma Em in UMR106 cells plated onto glass coverslips. The indicated concentrations of CGRP were applied successively (from lower to higher concentrations), with 2-min time interval (Int.), to the same coverslip of cells. This is one of many replicate experiments. The solid lines represent predicted baselines, which were periodically confirmed by treating coverslips with vehicle alone (as shown below). Throughout this study, the same scale is used for FI (arbitrary units), so that Em shifts are comparable. B, Negative controls: DW, EtOH, DMSO, or CT was independently applied to parallel coverslips of cells. Lower panel, The dose-response relationship between CGRP concentration and the change in Em (i.e. hyperpolarization). To quantify this relationship, observed decreases in FI (i.e. Em values), obtained at 180 sec after CGRP application, were plotted against the applied concentration of neuropeptide.

 
Compared with independent application to individual slides of cells, this method of cumulative application provides more reproducible data, and this general approach has been used for all the studies reported here. When we plot CGRP-induced hyperpolarization (measured after 180 sec of treatment) vs. CGRP concentration, we obtain a dose-dependent relationship, where the maximal effect is obtained at 100 nM (Fig. 1Go, lower panel). Assuming that the relationship between the changes in FI of DiBAC4(3) and the mV changes in Em are linear (47), the ED50 of CGRP is approximately 0.2 nM in these cells. In parallel experiments, we applied DW, EtOH (0.1%), DMSO (0.1%), or CT (100 nM) as negative controls; and we observed no appreciable changes (Fig. 1BGo).

Both Pina (1 µM), a specific opener of KATP channels, and forskolin (FSK; 0.5 µM), a specific activator of adenylate cyclase, induced similar membrane hyperpolarization in UMR106 cells. Figure 2Go shows the sensitivity of these hyperpolarization events to Glib (a specific inhibitor of KATP channels), TEA (an inhibitor of KCa channels), or H-89 (a selective inhibitor of cAMP-dependent kinases). As demonstrated (A), Glib (1 µM) blocked CGRP-induced hyperpolarization, TEA (1.5 mM) was without any effect, and H-89 (5 µM) produced only a modest attenuation of this CGRP effect. As Fig. 2BGo indicates, Pina-induced hyperpolarization is not identical to CGRP-induced changes in Em, and it typically consists of two major phases: a rapidly progressing initial phase that begins within 20 sec of application but then reverses to near-baseline value, followed by a somewhat larger but more gradual hyperpolarization that lasts for an extended period of time. Despite these differences in initial time-course, the overall effect produced by Pina at 180 sec is very similar to that produced by CGRP at the same time-point. Glib strongly inhibited the second phase of Pina-induced hyperpolarization and also significantly attenuated the acute phase. Either TEA or H-89 slightly attenuated Pina’s effect in both phases (B) but did not achieve statistical significance. Panel C shows that FSK produced a profile similar to that of Pina; however, this hyperpolarization was significantly attenuated or blocked by either TEA or H-89, respectively, and was not affected by Glib. Thus, the sensitivity of FSK-induced hyperpolarization to Glib, TEA, and H-89 clearly distinguished this effect from that produced by either CGRP or Pina. These relationships (at 3 min of treatment) are summarized in Fig. 3Go: the theme exhibited by these data are that Glib was very effective in blocking either CGRP- or Pina-induced hyperpolarization (but that it could not attenuate FSK-induced hyperpolarization) and that H-89 was markedly more effective in attenuating FSK-induced hyperpolarization than it was in limiting the action of CGRP.



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  		Figure 2. The effects of Glib, TEA, or H-89 on CGRP- (A), Pina- (B), or FSK-induced hyperpolarization (C) in adherent UMR106 cells. As indicated, cells were pretreated with either Glib (1 µM), TEA (1.5 mM), or H-89 (5 µM) for 10 min; and then CGRP (100 nM), Pina (1 µM), or FSK (0.5 µM) were applied to the cells.

 


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  		Figure 3. The inhibitory effects of Glib, TEA, or H-89 on the Em hyperpolarization produced by CGRP (upper), Pina (middle), or FSK action (lower) (summary of the data derived from Fig. 2Go). As before, the inhibition obtained at 3 min of hyperpolarization was plotted. a, P < 0.005; b, P < 0.001, vs. control.

 
Figure 4Go shows the interactions of CGRP, Pina, and FSK, in terms of their ability to alter the Em of UMR106 cells. After being exposed to the maximally effective concentration of CGRP (100 nM) for longer than 15 min, cells no longer would respond to Pina (1 µM), but they still responded to a subsequent treatment with FSK (0.5 µM) (A). Even after a long exposure to FSK (B), cells responded to CGRP (although the magnitude of this response was somewhat reduced, as was the case in the experiment depicted in panel A). A subsequent treatment with Pina, after this CGRP treatment, did not further induce hyperpolarization (B). As shown in panel C, pretreatment with Pina completely blocked the cells’ response to CGRP. In addition, no appreciable inhibitory interactions were observed between FSK and Pina (D and E).



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  		Figure 4. The interaction of CGRP, Pina, and/or FSK to alter Em in adherent UMR106 cells. As indicated by arrows, CGRP (100 nM), Pina (1 µM), or FSK (0.5 µM) was applied over the indicated period of time.

 
To investigate whether K+ is primarily involved in these rapid changes in Em, we next examined intracellular K+ mobilization. In PBFI-loaded UMR106 cell suspensions, the resting cellular level of [K+]i was estimated as 113.5 ± 18.7 mM (n = 60), which is very similar to the value previously reported by Civitelli et al. (47). We used cumulative applications of CGRP, as before, to demonstrate a dose-response relationship between CGRP applied and the decreases obtained in the [K+]i of UMR106 cells. The maximal effect was observed at 100 nM, after 3 min of treatment, and [K+]i was lowered by 51.9 ± 7.8 mM (n = 16), to roughly 60 mM (Fig. 5Go, upper). The dose-response relationship for K+ efflux was almost identical to that observed for altering Em (Fig. 2Go): CGRP (0.01–100 nM) dose-dependently decreased [K+]i, with an ED50 at approximately 0.2 nM. Typical time-courses demonstrating decreases in cellular [K+]i are shown in Fig. 6Go; the effect of CGRP, Pina, and FSK are shown as the initial lower trace in panels A, B, and C. With regard to negative controls, neither vehicle treatments (DW, EtOH, DMSO) nor CT produced any appreciable change in [K+]i (Fig. 5Go, lower) and were used to confirm a stable baseline in our experiments.



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  		Figure 5. Upper panel, The dose-response relationship between CGRP and decreases in [K+]i observed in suspended UMR106 cells. The same UMR106 cell suspension was treated with successively increasing concentrations of CGRP, essentially as described in the legend of Fig. 1Go (lower panel). The CGRP-induced decreases in [K+]i obtained after 180 sec of treatment (see Fig. 6Go) were measured and plotted directly against CGRP concentration to produce this dose-response curve. Lower panel, These negative controls were run repeatedly to confirm the predicted baselines in these experiments: DW, EtOH, DMSO, or CT were independently applied to parallel suspensions of UMR106 cells.

 


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  		Figure 6. The effects of Glib on CGRP- (A), Pina- (B), or FSK-induced (C) decreases in [K+]i in suspended UMR106 cells. As indicated on these traces, cells were initially treated with Glib (1 µM) before either CGRP (100 nM), Pina (1 µM), or FSK (0.5 µM) were applied to the cells. In addition, each of these experiments was also conducted in reverse, i.e. CGRP, Pina, or FSK were first applied to demonstrate the effect, and then Glib was applied to attempt to reverse the decrease in [K+]i (shown as the second set of traces in all three panels).

 
Figure 6Go provides pharmacological characterization of the CGRP-, Pina-, or FSK-induced changes in [K+]i in UMR106 cell suspensions. CGRP (100 nM) produced a gradual monophasic decline in [K+]i, just as it did for cellular Em. Both Pina (1 µM) and FSK (0.5 µM) also produce monophasic decreases in [K+]i more than 70% of the time (as shown here); but in a notable number of experiments, a biphasic response (composed of a short, slight acute phase followed by a larger and more gradual effect) was observed. The reason for this variation in the response of cells to Pina or FSK is not understood at the present time, but it seems to be independent of any additional pharmacological inhibitors.

Essentially as shown for the changes in Em demonstrated in Fig. 2Go, the CGRP-induced decrease in [K+]i was sensitive to Glib: after pretreatment with Glib (1 µM), cells were no longer sensitive to either CGRP or Pina (Fig. 6Go, A and B). Glib did appreciably attenuate FSK action, but this Glib effect did not achieve statistical significance in our experiments (panel C; also see Fig. 8Go). Interestingly, Glib reproducibly induced a strong reversal in the decrease in [K+]i observed in either CGRP- or Pina-treated cells (second half of the lower traces in panels A and B), whereas it elicited only small (but reproducible) reversals in either FSK-treated (lower trace, panel C) or untreated (upper traces in all panels) cells.



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  		Figure 8. The ability of Glib and TEA to attenuate or block the ability of CGRP, Pina, or FSK to decrease [K+]i in suspended UMR106 cells. Using time-courses similar to those presented in Fig. 7Go, the decrease in [K+]i, obtained 180 sec after application of the specified pharmacological agent, was measured and plotted, as shown. a, P < 0.001 vs. control.

 
Figure 7Go demonstrates the comparative sensitivities of CGRP, Pina, or FSK action to TEA. In good agreement with the data shown in Figs. 2Go and 3Go, the action of CGRP was slightly limited by TEA pretreatment, and similar attenuation was seen in the case of Pina action; however, the action of FSK was strongly blocked by TEA pretreatment. Figure 8Go further summarizes the ability of Glib or TEA to block these three agents (after 3 min of treatment). Both CGRP and Pina action were strongly blocked by Glib, whereas FSK action was attenuated only by TEA. Careful examination revealed that TEA produced weak attenuation of both the CGRP and Pina effects, but this 20–25% inhibition was only statistically significant in the case of CGRP action.



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  		Figure 7. The effects of TEA on CGRP-, Pina-, or FSK-induced decreases in [K+]i in suspended UMR106 cells. Cells were pretreated with TEA (1.5 mM) for 10 min and then treated with CGRP (100 nM), Pina (1 µM), or FSK (0.5 µM).

 
Figure 9Go shows the interactions of CGRP, Pina, and FSK on [K+]i in UMR106 cells. Cells treated with CGRP (100 nM) did not respond to Pina (1 µM), and conversely, Pina-treated cells did not respond to CGRP (A). On the other hand, cells treated with CGRP could still respond to FSK (0.5 µM), and FSK-treated cells could also respond to CGRP (B). Likewise, cells treated with Pina responded to FSK, and FSK-treated cells responded to Pina (data not shown). These data are essentially consistent with the results shown in Fig. 4Go, D and E, for changes produced in Em.



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  		Figure 9. The ability of CGRP, Pina, and FSK to interact in their influence of [K+]i in UMR106 cell suspensions. As indicated by arrows, CGRP (100 nM), Pina (1 µM), or FSK (0.5 µM) was applied, and after a 15-min Int., a second agent was applied to the cell suspension.

 
It has often been reported that a large K+ flux across the plasma membrane is accompanied by immediate changes in cell volume (50). Based on the data presented in Figs. 1–9GoGoGoGoGoGoGoGoGo, we were led to conduct microscopic analysis of UMR106 cells treated with CGRP. Figure 10Go presents our photomicroscopic analyses of the changes in cell shape and the shrinkage in cross-sectional cellular area produced within 10 min by 100 nM CGRP in adherent subconfluent cultures of UMR106 cells. As predicted from published reports, CGRP (100 nM) did rapidly induce changes in cell shape that began very soon after treatment and were most obvious after 10 min of treatment (C and D). When these shifts were analyzed by computerized 2-dimensional analysis of cellular size (cross-sectional area), CGRP (100 nM) significantly reduced the size of the cellular profile in a time-dependent manner, so that a 17% reduction in cross-sectional area was observed after 10 min of CGRP treatment (graph in lower panel, N = 20, paired Student’s t test, P < 0.01). Furthermore, pre- and cotreatment with Glib (1 µM) blocked the ability of CGRP to produce these changes in osteoblastic cell size or shape (E and F, and graph in lower panel,). Thus, the ability of CGRP to hyperpolarize Em and produce a strong K+ efflux seems to be accompanied by significant changes in cell morphology. As for all of CGRP’s pharmacological effects in these cells, these shifts in cellular morphology were most efficiently limited or blocked by Glib (TEA showed no effect).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In general, it is accepted that cellular Em depends on both the distribution and selective membrane permeabilities that are characteristic of several major cellular ions (51, 52). Furthermore, Em, itself, is known to directly regulate ion fluxes (e.g. attenuation or stimulation of Ca2+ influx through voltage-dependent channels) across the plasma membrane. In osteoblastic cells, it would be particularly important to understand Ca2+ use because the Ca2+ ion plays crucial roles not only in the regulation of cellular functions but also in the process of bone formation (i.e. biomineralization of matrix). Osteoblastic (UMR106) cells are known to possess several types of Ca2+ channels, among them: voltage-dependent Ca2+ (VDC) channels (53, 54) and Na+/Ca2+ exchangers (55), each of which can be involved in regulation of [Ca2+]i in response to various hormonal stimuli.

Pharmacological data from our previous studies (45, 46) suggested that CGRP initially binds to a CGRP-specific receptor and rapidly inhibits Ca2+ influx through VDC channels. The mechanism suggested by these data was rapid activation of KATP channels to alter Em. In the present study, we have directly demonstrated, in osteoblastic cells, that CGRP stimulates K+ efflux through a Glib-sensitive pathway, at the same time that it begins to induce membrane hyperpolarization. Based on the experiments reported herein and the observed sensitivities of specific compounds to pharmacological inhibitors, we suggest that Pina and CGRP employ a similar mechanism to produce their stimulation of potassium efflux and hyperpolarization of Em (we discuss some apparent differences below). These data are very consistent with our indirect pharmacological data (46) and again implicate that CGRP acts primarily on KATP channels. Thus, we believe that the inhibitory action of CGRP on Ca2+ uptake is primarily caused by KATP channel-mediated membrane hyperpolarization and subsequent inhibition of VDC channels.

With regard to their actions on K+ efflux, CGRP and Pina produce very similar overall decreases in cellular [K+]i, and CGRP is only marginally more sensitive to slight attenuation by TEA. With regard to the effects of CGRP or Pina to alter Em, there are strong general similarities and some notable minor differences. CGRP always produces a gradual monophasic hyperpolarization of Em, whereas Pina typically displays a biphasic effect. Although Pina and CGRP both seem to act through activation of the KATP channel, there is no reason that they must produce identical time courses (one compound is a 37-residue peptide that must first bind to a membrane receptor and activate cellular transduction pathways, whereas Pina is a smaller synthetic molecule that is thought to bind directly to a site on the pore-forming subunit of the KATP channel). Glib and Pina have long been known to oppose each other’s action on KATP channel activity. In the case of our experiment, Glib attenuated Pina’s initial phase in Em change by about 45% (measured at the peak), but it more strongly blocked (70% attenuation) the second (larger and more gradual) phase, producing effects very similar to the way in which it limits CGRP action in these experiments. This phenomenon, taken together with the data we have obtained from a limited number of preliminary experiments, suggests that another type of ion channel (quite possibly a Cl- channel) may serve as a secondary target for Pina and may strongly contribute to the acute-phase response to Pina. This interesting possibility should be further investigated (because Pina is described in the literature as a specific inhibitor of KATP channels, with no known secondary effects). However, these experiments have not been pursued as part of our present study because they will be extensive, and they digress (to a significant extent) from our present focus, which is the action of CGRP in its stimulation of osteoblastic cells.

A second way in which CGRP differs from Pina in these experiments concerns the fact that CGRP’s hyperpolarization of Em is, to some extent, attenuated by FSK pretreatment, whereas Pina action is unaffected by this pretreatment. It is generally thought that, in successive application of agents that share a common effector but employ different signaling pathways, the initial application of one agent may sometimes reduce the cellular responses that can be obtained by subsequent application of a second agent. However, we also observed, in these experiments, that CGRP action is slightly attenuated by the PKA inhibitor, H-89. In previous work (Kawase et al., unpublished observations), we have found, as have a number of other laboratories, that higher concentrations of CGRP (>=10 nM) are capable of stimulating modest increases in cellular cAMP production in UMR106 cells, so that 2- to 3-fold increases can be obtained in cellular levels within, roughly, 5 min. In addition to the central role of the KATP channels, therefore, we must also consider the possibility that CGRP induces a portion of its membrane hyperpolarization by simultaneously modulating other K+-transporting systems, such as activating KCa channels or even inhibiting Na+/K+-adenosine triphosphatase, perhaps by cAMP-dependent mechanism(s). In support of this possibility, Moreau et al. (43) recently demonstrated that cAMP activates KCa channels in the SaOS-2 and MG-63 osteoblastic cell-lines, and activation of these channels would produce membrane hyperpolarization. In agreement with these published data, we also have generated data (Kawase et al., manuscript in preparation) that strongly suggest that FSK activates KCa channels in UMR106 cells (such activation was unaffected by Glib, but completely blocked by TEA), without affecting KATP channels (in these as-yet-unpublished studies, the effects of Pina and FSK were completely independent and distinguishable). Recent published electrophysiological data from Moreau et al. (44) indicate that KCa and KATP channels are the primary modulators of K+ flux in osteoblastic cells under normal circumstances. Because we currently do not understand what contribution KCa channel activation (which seems small) makes, with respect to the contribution of KATP channel activation (which seems large) to the overall stimulation of K+ efflux and Em hyperpolarization by CGRP, we are now turning specifically to examine how large a role KCa channel activation can play in CGRP’s osteoblastic action.

As to the question of CGRP specificity (i.e. binding site), we have once again confirmed that rat CT (1–100 nM) showed no significant effects, either on Em or [K+]i, in UMR106 cells. Furthermore, the ED50 value for CGRP actions, calculated both from Em and [K+]i data, is estimated as approximately 0.2 nM, which is very consistent with the low KD value calculated from our previous rat 125I-CGRP binding assays (Burns et al., unpublished observations). Therefore, CGRP, most probably, binds to authentic high-affinity CGRP receptors on osteoblasts to produce these effects; to our knowledge, these high-affinity osteoblastic CGRP receptors have not yet been isolated.

It frequently has been reported that KATP and/or other K+ channels are involved in the regulation of cell size and cell volume in a variety of cell types (50, 56). Additional recent reports also propose that activation of K+ channels directly or indirectly trigger cell proliferation and/or cell differentiation by causing cell shrinkage and initiating shifts in cell morphology (57, 58, 59). Clearly, this is an area of cell biology that remains to be fully understood; however, we did observe significant shrinkage in measurable cell area, and we noticed obvious retraction of cell cytoplasm after CGRP stimulation of UMR106 cells. Furthermore, the mechanism for these cellular changes was Glib-sensitive, once again implicating the primary involvement of KATP channels. As stated before, accumulated evidence suggests that CGRP is intimately involved in controlling many aspects of bone formation and is capable of acting as some type of an anabolic factor; but little is known, so far, about how it actually promotes the activation of osteoblasts. In preliminary studies (e.g. Ref. 31), we have found that both short and extended treatments with CGRP, or with Pina, stimulate expression of certain phenotypically important osteoblastic genes that are involved in the differentiation and maturation of functional osteoblasts [e.g. Col I ({alpha} 1), bone sialoprotein, osteopontin, and the osteoblast-specific transcription factor CBFA-1]. This up-regulation of functional osteoblastic markers is, in fact, paralleled by increased osteoblastic activity: chronic treatment with as low as 4 nM of CGRP more effectively promotes biomineralization in confluent primary cultures of rat osteoblasts than does PTH (31). In light of these and other data, we propose that CGRP acts directly on osteoblasts to change Em and produce cell shrinkage; these changes might play important roles in triggering activation of osteoblastic phenotypic function.

There has been very little information available about the pathophysiological roles of K+ channels (and resulting changes in Em) in osteoblastic functions. However, Moreau et al. (44) have very recently demonstrated (with pharmacological, biochemical, and electrophysiological data) that osteoblastic cells possess both KATP and KCa channels and that these two K+ channels are cooperatively involved in the regulation of osteocalcin secretion. Taken together with our present findings, those results raise the strong possibility that changes in Em directly act as triggers for certain cellular functions, even in nonexcitable cells such as osteoblasts. Further studies are necessary to more fully explore this intriguing postulate.

In both our previous (45, 46) and present studies, ion fluxes and Em measurements have been made on large populations of osteoblastic cells, but no experiments have been performed, so far, with single cells or preparations of single channels. To more carefully characterize and identify the subtypes of KATP channels activated by CGRP in osteoblasts and to more fully understand the relationship between K+ channel activation and cellular Ca2+ use, we now need to turn more heavily toward an approach that will allow us to study combinations of single adherent cells. In particular, an electrophysiological approach is now needed to complement the molecular biology data that we are developing.


   Footnotes
 
1 This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, and Science of Japan and a Merit Review from the United States Department of Veterans Affairs. Back

Received November 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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