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Departments of Pharmacology and of Medicine, University of Pittsburgh School of Medicine (P.A.F.), Pittsburgh, Pennsylvania 15261; the Department of Pharmacology and Toxicology, Dartmouth Medical School (F.A.G.), Hanover, New Hampshire 03755; and the Institute for Biological Sciences, National Research Council of Canada (P.M., J.F.W., G.E.W.), Ottawa, Ontario, Canada K1A OR6
Address all correspondence and requests for reprints to: Peter A. Friedman, Ph.D., Department of Pharmacology, University of Pittsburgh School of Medicine, E-1347 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261. E-mail: PAF10{at}pitt.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Molecular cloning of the PTH receptor established three important points. First, PTH and PTHrP bind to the receptor, referred to as the type I receptor, with comparable affinity; second, as noted above, the PTH/PTHrP receptor couples, or at least is able to stimulate, adenylyl cyclase and PI-PLC (3); and third, both signaling events occur through a common receptor (4).
Nevertheless, some findings cannot be easily accommodated by the idea that all PTH effects are mediated by a canonical signaling mechanism or by a single receptor. For example, in some instances PTH activates adenylyl cyclase but not PI-PLC (5, 6, 7), whereas in others PTH activates PI-PLC but not adenylyl cyclase (8, 9, 10, 11). Second, carboxyl-terminal PTH fragments are biologically active but do not act through the type I PTH/PTHrP receptor (12, 13, 14, 15). These findings suggest that cell-specific modifications of the receptor or downstream events involved in receptor signaling are responsible for these disparities.
In the kidneys, PTH receptors are prominently expressed on proximal and
distal tubules (16, 17). However, activation of proximal and distal PTH
receptors has strikingly different actions. In proximal tubules, PTH
exerts a host of biochemical effects including activation of
25-hydroxyvitamin D-1
-hydroxylase, gluconeogenesis, and
ammoniagenesis and inhibition of Na+-phosphate absorption,
Na+/H+ exchange, and
Na+/Ca2+ exchange. All of these effects may
involve calcium signaling, characterized by a transient rise in
intracellular calcium ([Ca2+]i) and
1,4,5-trisphosphate (Ins[1,4,5]P3) formation. The
PTH-induced elevation of [Ca2+]i in proximal
cells is due entirely to release from intracellular stores; neither
removal of extracellular calcium nor treatment with a calcium channel
blocker inhibits the transient rise in
[Ca2+]i. Furthermore, PTH does not affect net
cellular calcium absorption by proximal nephrons. By contrast, in
distal tubules, PTH potently stimulates transcellular calcium
absorption that is accompanied by a slow and sustained rise in
[Ca2+]i, but does not stimulate PI-PLC
hydrolysis or cause Ins[1,4,5]P3 accumulation. The rise
in [Ca2+]i in distal tubule cells is
dependent entirely on extracellular calcium. Addition of a calcium
channel blocker (18) or removal of extracellular calcium (19) abolishes
the sustained PTH-stimulated rise in [Ca2+]i.
In contrast to the prompt transient rise in
[Ca2+]i in proximal tubule cells, the
sustained increase in [Ca2+]i in distal
tubule cells begins after a latency of 8–10 min (18, 19). The origin
of this delay is uncertain, but appears to involve protein translation
because it is inhibited by cycloheximide (20). These features might
suggest that PTH-stimulated calcium transport by distal cells does not
require protein kinase C (PKC) activation. However, we established that
activation of both PKC and protein kinase A (PKA) is necessary for the
stimulatory effect (21).
PKC is activated by diacylglycerol (DAG), which is produced consequent to receptor-mediated hydrolysis of phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (22). It was initially assumed that DAG involved the action of PLC coupled to the breakdown of PIP2. It is now recognized that DAG may also arise from the activation of phospholipase D (PLD) and the ensuing hydrolysis of phosphatidylcholine to form phosphatidic acid, which is converted to DAG by the action of phosphatidic acid phosphohydrolase, and choline. DAG so formed may be responsible for activation of calcium-independent forms of PKC (23).
The sequence within PTH responsible for activating PKC has been suggested to be comprised of residues 29–32 (13, 14) and has been referred to as the PKC-activating domain of PTH. Amino-terminal PTH analogs shorter than 32 residues failed to activate PKC in rat osteosarcoma (ROS) cells (13, 14, 24).
Notably, although PKC activation was mandatory for the action of PTH on distal tubule cells (21), the findings suggested that stimulation of transport proceeded through a pathway that did not involve hydrolysis of phosphatidylinositol 4,5-bisphosphate, because measurable accumulation of Ins[1,4,5]P3 did not occur in distal tubule cells after challenge with PTH-(1–34). Comparable experiments on proximal tubule cells, in which PTH inhibits Na+-dependent phosphate absorption, revealed Ins[1,4,5]P3 formation (21).
In the present investigation we took advantage of the presence within the kidney of the two cell types (proximal and distal tubule cells) exhibiting distinct patterns of PTH receptor signaling to characterize the origin of the differential effects of ligand occupancy. For these studies we employed synthetic PTH analogs to test the hypothesis that stimulation of calcium transport and activation of PKC in distal tubule cells are mediated by a PLCß-independent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary cultures of mouse proximal and distal tubule2 cells were also used. These were isolated by a double antibody immunodissection procedure (28). Primary cultures were grown under the same conditions as the cell lines, but with 10% FCS (Sigma Chemical Co.). Cells were placed in serum-free DMEM/Ham’s F-12 medium 16 h before use.
PTH fragments
Carboxyl-terminal amide fragments of human PTH or PTHrP, except
where noted [PTH-(1–27)NH2, PTH-(1–28)NH2,
PTH-(1–29)NH2, PTH-(1–30)NH2,
PTH-(1–31)NH2, PTH-(1–34)NH2,
PTHrP-(1–31)NH2, PTH-(1–34)NH2, and
PTH-(3–34)], and PTHrP analogs [PTHrP-(1–31)NH2 and
PTHrP-(1–34)NH2] were synthesized using the
9-fluorenylmethoxycarbonyl (F-moc) procedure (29) and a continuous flow
peptide synthesizer (model 9050, PerSeptive Biosystems, Framingham, MA)
as described previously (30).
Intracellular calcium
[Ca2+]i was measured in single cells
or clusters of up to three cells as detailed previously (27). In brief,
proximal or distal convoluted tubule cells plated on coverslips were
incubated for 60 min at 37 C with 10 µM fura-2/AM
(Molecular Probes, Inc., Eugene, OR) in a buffer
consisting of 140 mM Na+, 148 mM
Cl-, 5 mM K+, 1 mM
Ca2+, 1 mM Mg2+, 28 mM
HEPES, and 18 mM Tris-HCl with 10 mM glucose at
pH 7.4 and adjusted to 295 mosmol. The cells were then rinsed several
times, placed in a temperature-controlled incubator (Narishige Medical
Systems Corp., Greenvale, NY), and mounted on the stage of an inverted
microscope (Nikon Diaphot, Nikon, Melville,
NY). Emitted fluorescence was measured with a Nikon
Photoscan-2. Calibration was performed as previously described
(27).
45Ca2+ uptake
Calcium uptake was measured by a rapid filtration technique
(27). Five to 6 x 106 cells/60-mm dish were removed
by brief (
5 min) treatment with 0.125% trypsin and rinsed with a
modified Krebs-Ringer solution containing 140 mM NaCl, 5
mM KCl, 1 mM CaCl2, and 1
mM MgCl2 buffered with 18 mM Tris
base and 28 mM HEPES to pH 7.40 ± 0.01 (to 295
± 1 mosmol). Buffer, with or without PTH or other test drugs (total
volume, 100 µl), was placed in 12 x 75-mm polystyrene tubes. A
50-µl aliquot of cells was added for 1 min before addition and rapid
mixing of 50 µl 45Ca2+ to initiate isotope
uptake. Sample tubes were maintained at 37 C in a shaking incubator.
Tracer uptake was terminated after 1 min by rapid addition of ice-cold
isoosmotic, Li2SO4-HEPES rinse buffer [140
mM Li2SO4 and 10 mM
HEPES (pH 7.40); 295 ± 2 mosmol] and filtered onto
Whatman GF/C filters (Clifton, NJ) using a Millipore Corp. 12-port manifold (Bedford, MA) followed by two additional
rinses with Li2SO4-HEPES buffer. Nonspecific
binding to filters and cells was determined in the presence of
Li2SO4-HEPES buffer added to the cells before
the addition of 45Ca2+. Calcium uptake was
normalized for protein content, which was measured using the Lowry
procedure on 50-µl aliquots of cells (31). Tracer uptakes are
expressed as nanomoles per min/mg protein.
cAMP
Cells were trypsinized, washed, and resuspended in buffer 135
mM NaCl, 4 mM KCl, 1.0 mM
KH2PO4, 1.2 mM CaCl2,
1.2 mM MgCl2, 10 mM HEPES, and 5
mM glucose) to a final concentration of 1 x
106 cells/ml. A 50-µl aliquot of the cell suspension was
taken for protein determination. A second 50-µl aliquot was added to
a prewarmed plastic tube of 50 µl cAMP buffer containing 0.4% BSA
and 0.2 mM rolipram in the presence or absence of the
indicated PTH analog. After 15 min in a 37 C shaking water bath, 1 ml
ice-cold 0.132 M trichloroacetic acid was added, and
samples were vortexed and stored at -70 C. For measurement of cAMP,
the tubes were thawed and centrifuged at 1200 x g for
30 min, and 0.9 ml supernatant was removed and transferred to a glass
tube. The sample was extracted twice with 0.9 ml
H2O-saturated ether, and the final aqueous solution was
dried. cAMP was measured by RIA using a kit from Diagnostic Products Corp. (Los Angeles, CA).
Inositol trisphosphate and DAG
Ins[1,4,5]P3 was measured by RIA. Primary cultures
of proximal tubule cells were grown to confluence in six-well plates.
Cells were serum and antibiotic starved overnight before treatment with
PTH. Cells were rinsed with a buffer composed of 140 mM
NaCl, 4.6 mM KCl, 1 mM CaCl2, and 1
mM MgCl2 adjusted to pH 7.40 ± 0.01 with
28 mM HEPES and 18 mM Tris base (to 295 ±
2 mosmol). A second rinse used a buffer containing 50 mM
LiCl, which isoosmotically replaced NaCl. Cells were treated in the
wells with a total of 500 µl buffer with or without PTH for 2 min at
37 C on a shaking incubator. The treatment was terminated by the
addition of 100 µl ice-cold 20% perchloric acid. Wells were scraped,
and the contents were placed in a 1.5-ml conical centrifuge tube and
pelleted by centrifugation at 2000 x g for 15 min at 4
C. The supernatant was neutralized to approximately pH 7.5 by the
addition of 175 µl buffer consisting of 1.5 M KOH and 120
mM HEPES. The mixture was centrifuged as described above to
remove KClO4 precipitate. A 500-µl aliquot of the
supernatant was removed and placed in a 1.5-ml conical centrifuge tube.
Samples were evaporated to dryness at medium heat (43 C) with a
Speed-Vac (Savant Instruments, Farmingdale, NY) and reconstituted in
100 µl distilled water. Samples and standards (100 µl) were
analyzed with an [3H]InsP3 assay kit
(Amersham, Arlington Heights, IL). Protein-bound samples
and standards were placed in 5 ml scintillation fluid and counted by
ß-emission spectrometry.
DAG accumulation was determined using the method developed by Griendling et al. (32) and modified as described previously (33). Distal convoluted tubule cells were grown to 90% confluence on 100-mm dishes as described above and labeled with [3H]arachidonic acid (New England Nuclear Corp., Boston, MA) for 4 h. PTH was added for the times shown in Results. The experiment was terminated, and DAG was extracted and analyzed by TLC as detailed previously (33).
PLD
PLD was assayed by exploiting its unique ability to catalyze the
transphosphatidylation of a primary electrophile, butanol in this case.
Distal convoluted tubule cells were grown to confluence on 35-mm
dishes. At the time of the experiment, 20 µCi
[4-N-3H]butanol (ARC, St. Louis, MO) were added to each
dish, with or without 100 nM PTH-(1–84), for 30 min at 37
C. The reaction was stopped by adding 0.9 ml ice-cold 0.6 N
perchloric acid. The cells were scraped in to a 15-ml centrifuge
tube, resuspended in 1 ml buffer A
(CHCl3-CH3CHCOOH, 100:0.5) and applied to
500-mg, 6-cc silica Sep-Pak columns (Waters, Milford, MA) that had been
preequilibrated with 10 ml CHCl3. The column was washed
with 6 ml buffer A, and the eluate was discarded. Phospholipids were
eluted with 5 ml buffer C (CHCl3-MeOH-H2O,
2:1:0.8), evaporated to dryness in a Speed-Vac, and resuspended in 100
µl CHCl3-MeOH (4:1) for analysis by TLC.
Phosphatidylbutanol was resolved by short bed, continuous development
TLC, using silica gel G-precoated, channeled plates (Analtech, Newark,
DE) and a mobile phase consisting of
CHCl3-MeOH-CH3COOH (65:15:2) as described by
Sarri et al. (34). A phosphatidylbutanol standard
(1,2-dimyristoyl-sn-glycero-3-phosphobutanol; Avanti Polar
Lipids, Alabaster, AL) was run in parallel. Identification was
performed with iodine. Samples were cut from the poly-backed plate and
counted by ß-scintillation spectrometry.
Materials
U73122 and U73343 were purchased from Research Biochemicals International (Natick, MA). Rolipram was purchased
from Biomol Research Laboratories, Inc. (Plymouth Meeting,
PA). Human PTH-(19–84) was provided by Dr. Harald Jüppner,
Massachusetts General Hospital (Boston, MA).
45Ca2+ (1 µCi/ml; carrier-free) was obtained
from New England Nuclear (Boston, MA). Chemicals and other reagents
were of the highest grade commercially available. Solutions containing
drugs were prepared fresh daily.
Statistical analysis
The effects of experimental treatments were assessed by paired
comparisons within experiments and are reported as the mean ±
SE of n independent experiments. Comparisons between
control and experimental treatment groups were evaluated by Student’s
t test (Instat, GraphPad Software, Inc., San
Diego, CA). P 0.05 was assumed to be significant.
Kinetic parameters and curve fitting were performed with Prism software
(GraphPad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, PTH-(1–34) increased
[Ca2+]i by 200 nM, similar to the
effects reported previously with the comparable bovine analog (18).
PTH-(1–31) also increased [Ca2+]i, although
to a somewhat lesser extent. Figure 1b shows a complete dose-response
curve for PTH-(1–31). The maximal sustained increase in
[Ca2+]i elicited by PTH-(1–34) (330 ±
5 nM; n = 4) was comparable to that caused by bovine
PTH-(1–34) (18), but was greater than that caused by PTH-(1–31)
(275 ± 4; n = 6). The smaller maximal effect, an index of
intrinsic activity, is consistent with the view that in the distal
tubule, PTH-(1–31) is a partial receptor agonist.
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. We
also examined the action of the carboxyl-terminal peptide,
PTH-(19–84). It had no detectable effect on
[Ca2+]i in distal convoluted tubule cells
(data not shown).
If a common PTH/PTHrP receptor mediates PTH actions in distal tubules,
then the receptor antagonist PTH-(7–34) should block the rise in
[Ca2+]i. We tested this postulate by
analyzing the effects of PTH-(7–34) on changes in
[Ca2+]i induced by concentrations of
PTH-(1–34) and PTH-(1–31) that elicited half-maximal increases in
[Ca2+]i (10-10 M).
The results, shown in Fig. 2, revealed
that intermediate concentrations of PTH-(7–34) (10-9
M) caused partial suppression of PTH-(1–34)-stimulated
increases in [Ca2+]i; higher concentrations
(10-8 M) abolished the effects of PTH-(1–34)
or PTH-(1–31).
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, PTH-(1–31) had no measurable on
[Ca2+]i in a single proximal tubule
S1 cell. Equivalent results were obtained in four
paired independent experiments (Fig. 3b). The absence of an effect of
PTH-(1–31) on [Ca2+]i in proximal tubule
cells contrasts with its prominent action on distal convoluted tubule
cells (Fig. 1, a–c). The spiking rise in
[Ca2+]i that attends PTH action in proximal
tubule cells is primarily due to the release of calcium from
intracellular stores, which is secondary to activation of PLCß.
Hence, the failure of PTH-(1–31) to elevate
[Ca2+]i in proximal tubule cells is
consistent with the view that in these cells PTH-(1–31) does not
activate PLCß, as originally proposed by Whitfield et al.
(13, 14). PTH-(19–84) had no detectable effect on
[Ca2+]i in proximal tubule cells (data not
shown). This latter observation suggests that the 29–32 region is not
sufficient to activate PLCß and that amino-terminal sequence is
required.
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shows that U73122 virtually abolished
the PTH-(1–34)-stimulated rise in [Ca2+]i in
a proximal tubule cell, but has no detectable effect of its own. The
inactive analog, U73343, had no effect on resting or PTH-stimulated
changes in [Ca2+]i. Likewise, U73122
inhibited PTH-stimulated Ins[1,4,5]P3 formation in
proximal tubule cells (Fig. 4b). These findings fortify the conclusion
that the effects of PTH on [Ca2+]i in
proximal tubule cells are mediated by PLCß. We also determined the
effects of U73122 on PTH-dependent calcium uptake by distal tubule
cells. In prior work we established the ability of distal cells to
mount an appropriate PLC-ß response, characterized by a transient
rise in calcium and inositol trisphosphate (IP3) formation
(38). Moreover, U73122 abolished the rise in both calcium and
IP3 formation. In the present studies we used the same
concentration of U73122 (10 µM). As shown in Table 1, inhibition of PLCß with U73122 under
conditions identical to those that blocked hormone-stimulated increases
in [Ca2+]i and Ins[1,4,5]P3
formation in proximal tubule cells had no effect on
45Ca2+ uptake by distal tubule cells.
Predictably, U73122 had no detectable effect on PTH-(1–34)- or
PTH-(1–31)-induced increases in [Ca2+]i in
distal tubule cells (data not shown).
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, although both analogs
raised [Ca2+]i, PTH-(1–34) was more
efficacious that its 1–31 counterpart, whereas the two PTHrP fragments
exhibited comparable activities. These findings support the view that
the 29–32 sequence of PTH is not obligatorily or uniquely associated
with activation of PLCß, but may activate PKC through other
pathways.
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shows that in six paired independent
comparisons, PTH-(1–84) increased PLD activity (P <
0.01). Hydrolysis of phosphatidylcholine by PLD releases phosphatidic
acid that, in turn, is converted to DAG by the action of phosphatidic
acid phosphohydrolase. Thus, if PTH acts through PLD in distal
convoluted tubule cells, it should stimulate DAG formation despite the
absence of Ins[1,4,5]P3 accumulation. As illustrated in
Fig. 7, PTH caused a time-dependent
stimulation of DAG by distal convoluted tubule cells. Maximal
activation occurred at 2 min, a time appreciably before PTH increases
calcium entry by these cells (Fig. 1a). We further tested the postulate
that stimulation of PKC by PTH proceeds through a PLCß-independent
mechanism in distal convoluted tubule cells by analyzing the effects of
U73122. The results, shown in Table 2,
confirm that doses of U73122 that inhibit PTH-stimulated
Ins[1,4,5]P3 accumulation by proximal cells (Fig. 4b)
(21) or -adrenergically induced Ins[1,4,5]P3 formation
in distal cells (38) did not suppress activation of PKC.
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, a and
b). Such an effect implies that in proximal tubule cells the rise in
[Ca2+]i does not require activation of
adenylyl cyclase, which depends upon the presence of Ser and Ala in
positions 1 and 2 (40). To test explicitly the hypothesis that the rise
in [Ca2+]i in proximal tubule cells is
mediated through a cAMP-independent mechanism, we examined the effects
of PTH-(3–34). In seven paired studies, resting
[Ca2+]i averaged 99 ± 4 nM
and increased by 33% to 132 ± 5 nM
(P < 0.01). Nonetheless, the magnitude of this change
was less than that elicited by PTH-(1–34) (change in
[Ca2+]i, 116 ± 4) or PTH-(1–84).
To learn whether the origin of the dissimilar actions of the PTH
analogs, particularly with respect to PTH-(1–31), which increased
[Ca2+]i in distal tubule cells but not in
proximal tubule cells, was attributable to differences in the
generation of second messengers, we analyzed the effects of selected
amino-terminal PTH analogs on cAMP formation. The results, shown in
Fig. 8, revealed distinct differences
among the various analogs, although in all circumstances their effects
on cAMP formation in distal convoluted tubule and primary cultures of
proximal tubule cells were comparable. PTH-(1–30) failed to stimulate
cAMP accumulation in either distal tubule or proximal tubule cells,
whereas PTH-(1–31) caused nearly a 20-fold increase in cAMP formation
in both distal tubule and proximal tubule cells. Thus, although
PTH-(1–31) induced comparable cAMP formation in proximal tubule cells,
it failed to increase [Ca2+]i. These findings
suggest that the rise in [Ca2+]i in proximal
tubule cells cannot be triggered solely by a cAMP- or PKC-dependent
mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both PTH-(1–31) and PTH-(1–34) caused comparable accumulation of cAMP by proximal and distal convoluted tubule cells. Thus, it is unlikely that preferential activation of adenylyl cyclase accounts for the observed differences in calcium entry. PTH-(1–27), PTH-(1–28), PTH-(1–29), and PTH-(1–30) failed to stimulate detectable cAMP formation in proximal and distal convoluted tubule cells. The findings with distal tubule cells differ somewhat from the pattern of adenylyl cyclase stimulation by these analogs in rat osteosarcoma 17/2 cells, where PTH-(1–30)NH2 was nearly equally effective as PTH-(1–31) and PTH-(1–30) (30). The divergence in the results may be due to species differences as well as to differences in PTH/PTHrP receptor number. For instance, when the human type I PTH/PTHrP receptor was expressed in LLC-PK1 cells, PTH-(1–34), PTH-(1–31), and PTH-(1–30) caused equivalent stimulation of adenylyl cyclase, with identical half-maximal concentrations. Likewise, all three PTH analogs elicited comparable InsP3 formation and rise in [Ca2+]i. However, when equivalent numbers of the rat type I PTH/PTHrP receptors were expressed in LLC-PK1 cells, PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) was an order of magnitude less potent in activating adenylyl cyclase and failed to stimulate PLCß, as evidenced by the absence of InsP3 formation (41).
Although it could be argued that proximal and distal convoluted tubule cells express different PTH receptor isoforms, we think this is unlikely for the following reasons. First, at present there is scant evidence that alternatively spliced isoforms of the type I PTH/PTHrP receptor are functionally expressed (42, 43). Second, a PTH/PTHrP receptor cloned from mouse kidney is indistinguishable from that in the distal convoluted tubule (44, 45). Third, the EC50 values for increasing [Ca2+]i by PTH-(1–34) and PTH-(1–31) in distal tubule cells were indistinguishable: 2.3 vs. 2.9 x 10-10 M, respectively. Likewise, the EC50 values for stimulation of adenylyl cyclase by PTH-(1–84) in proximal and distal convoluted tubule cells were comparable (41 and 24 nM, respectively; calculated from data in Ref. 21). The Hill coefficient of 1 derived from the dose-response curves for PTH-(1–34) and PTH-(1–31) is also compatible with a single class of receptor. Fourth, PTH-(7–34) blocked PTH-(1–34)- and PTH-(1–31)-induced increases in [Ca2+]i, suggesting that both peptides activate the classical type I PTH/PTHrP receptor. The finding that PTH-(3–34) (13, 46) and PTH-(7–34) (Jouishomme, H., unpublished observations) activated PKC but evoked only a limited rise in [Ca2+]i in proximal tubule cells and failed to increase [Ca2+]i in distal tubule cells suggests that cAMP is needed to trigger the rise in [Ca2+]i or that the first two amino acids independently are important for maximizing the intrinsic activity of PLCß. Alternatively, the partial increase in [Ca2+]i induced by PTH-(3–34) and PTH-(7–34) may result from a failure of these analogs to stimulate InsP3 formation maximally.
The fact that the 1–34 and 1–31 analogs of PTH and PTHrP elicited similar effects in distal convoluted tubule cells is consistent with the idea that the PTH receptor mediating these actions recognized both PTH and PTHrP, as demonstrated for the type I PTH/PTHrP receptor (47). Clearly, certain PTH analogs working through a common type I PTH/PTHrP receptor activate adenylyl cyclase in some target cells whereas others do not. It is possible that this may be a consequence of the facility with which these analogs are able to induce conformational changes in the receptor.
The involvement of PLCß in transducing the action of PTH in proximal and distal tubule cells was assessed with the PLCß inhibitor, U73122. This compound abolished PTH-induced transient rises in [Ca2+]i in proximal convoluted tubule cells and InsP3 formation, but did not suppress PTH-stimulated 45Ca2+ uptake by distal convoluted tubule cells. In earlier studies (21) it was established that PTH-stimulated calcium entry in distal tubule cells required activation of both PKA and PKC. However, although PKC participation was necessary, its activation apparently proceeds through a PLCß-independent pathway, as PTH did not cause measurable InsP3 formation. It is possible, however, that large doses of PTH-(1–30) may activate PLCß in cells expressing high numbers of the human PTH/PTHrP receptor, where the Km for adenylyl cyclase is similar for PTH-(1–34), PTH-(1–31), and PTH-(1–30) (40). Nonetheless, stimulation of calcium uptake and the rise in [Ca2+]i in distal convoluted tubule cells require activation of PKC (21). The results with U73122 support the view that stimulation of PKC by PTH in distal convoluted tubule cells does not involve PLCß. Therefore, we surmise that type I PTH/PTHrP receptors are capable of activating phospholipases other than PLCß and that the structural requirements for such activation differ from the 29–32 sequence that is necessary for activation of PLCß in ROS 17/2 osteosarcoma cells (13, 14).
An attractive candidate for such a non-PLCß mechanism is PLD, which
hydrolyzes membrane phosphatidylcholine and indirectly generates
PKC-activating diacylglycerols without the attendant formation of
InsP3 and associated mobilization of intracellular
Ca2+ (48). The results depicted in Fig. 6 show that PTH
caused a moderate, but consistent, activation of PLD. The observation
that PTH also induced DAG formation serves as an independent
confirmation of PLD activation and the fact that DAG accumulation
proceeds in the absence of InsP3. The increase in PLD
activity was relatively modest, especially in the face of a comparable
increase in DAG formation. These findings suggest that a relatively
large pool of the DAG precursor, phosphatidic acid, or the presence of
additional routes of DAG formation may exist. In this regard, it is
known that DAG formation can arise from phospholipase A2
hydrolysis of phosphatidylcholine and that PTH activates
PLA2 in some renal cells (49, 50, 51). A recent study likewise
reported that PTH activates PLD in osteoblast-like UMR-106 cells (52).
At present, the mechanism by which PTH receptors activate PLD is
unclear. It is currently thought that PLD is activated by monomeric
(small) G proteins or PKC (47, 53). The identities of these mediators
remain to be identified. It is uncertain how or why the type I
PTH/PTHrP receptor preferentially activates PLD, because distal cells
express PLCß that can be activated through a Gq/11
mechanism (38).
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Acknowledgments |
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Footnotes |
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2 The terms distal tubule and proximal tubule
cells denote the mixed populations of cortical ascending limb and
distal convoluted tubule cells, or S1 and S2 proximal tubule cells,
respectively, that are isolated by immunoselection (28 ) and grown in
primary cell culture. In contrast, distal convoluted tubule cells and
proximal convoluted tubule cells refer to immortalized lines of clonal
distal and proximal convoluted tubule cells.
Received May 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Adrenergic receptors
activate phosholipase C in renal epithelial cells. Mol Pharmacol 50:407–414[Abstract]
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  T. de Groot, K. Lee, M. Langeslag, Q. Xi, K. Jalink, R. J.M. Bindels, and J. G.J. Hoenderop Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation J. Am. Soc. Nephrol., August 1, 2009; 20(8): 1693 - 1704. [Abstract] [Full Text] [PDF]
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 Y.-L. Zhang, J. A. Frangos, and M. Chachisvilis Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1391 - C1399. [Abstract] [Full Text] [PDF]
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 B. Wang, Y. Yang, and P. A. Friedman Na/H Exchange Regulatory Factor 1, a Novel AKT-associating Protein, Regulates Extracellular Signal-regulated Kinase Signaling through a B-Raf-Mediated Pathway Mol. Biol. Cell, April 1, 2008; 19(4): 1637 - 1645. [Abstract] [Full Text] [PDF]
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 M. J. Mahon, T. M. Bonacci, P. Divieti, and A. V. Smrcka A Docking Site for G Protein {beta}{gamma} Subunits on the Parathyroid Hormone 1 Receptor Supports Signaling through Multiple Pathways Mol. Endocrinol., January 1, 2006; 20(1): 136 - 146. [Abstract] [Full Text] [PDF]
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 R. Cunningham, X. E, D. Steplock, S. Shenolikar, and E. J. Weinman Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1-/- renal proximal tubule cells and wild-type cells adapted to low-phosphate media Am J Physiol Renal Physiol, October 1, 2005; 289(4): F933 - F938. [Abstract] [Full Text] [PDF]
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 T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
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 W. B. Sneddon, C. E. Magyar, G. E. Willick, C. A. Syme, F. Galbiati, A. Bisello, and P. A. Friedman Ligand-Selective Dissociation of Activation and Internalization of the Parathyroid Hormone (PTH) Receptor: Conditional Efficacy of PTH Peptide Fragments Endocrinology, June 1, 2004; 145(6): 2815 - 2823. [Abstract] [Full Text] [PDF]
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J. Ba, D. Brown, and P. A. Friedman Calcium-sensing receptor regulation of PTH-inhibitable proximal tubule phosphate transport Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1233 - F1243. [Abstract] [Full Text]
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M. J. Mahon, J. A. Cole, E. D. Lederer, and G. V. Segre Na+/H+ Exchanger-Regulatory Factor 1 Mediates Inhibition of Phosphate Transport by Parathyroid Hormone and Second Messengers by Acting at Multiple Sites in Opossum Kidney Cells Mol. Endocrinol., November 1, 2003; 17(11): 2355 - 2364. [Abstract] [Full Text] [PDF]
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 W. B. Sneddon, C. A. Syme, A. Bisello, C. E. Magyar, M. D. Rochdi, J.-L. Parent, E. J. Weinman, A. B. Abou-Samra, and P. A. Friedman Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50) J. Biol. Chem., October 31, 2003; 278(44): 43787 - 43796. [Abstract] [Full Text] [PDF]
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M. Shimada, X. Chen, T. Cvrk, H. Hilfiker, M. Parfenova, and G. V. Segre Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide J. Biol. Chem., August 23, 2002; 277(35): 31774 - 31780. [Abstract] [Full Text] [PDF]
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 H. A. W. Tawfeek, J. Che, F. Qian, and A. B. Abou-Samra Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E545 - E557. [Abstract] [Full Text] [PDF]
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A. ValIn, C. Guillen, and P. Esbrit C-Terminal Parathyroid Hormone-Related Protein (PTHrP) (107-139) Stimulates Intracellular Ca2+ through a Receptor Different from the Type 1 PTH/PTHrP Receptor in Osteoblastic Osteosarcoma UMR 106 Cells Endocrinology, July 1, 2001; 142(7): 2752 - 2759. [Abstract] [Full Text] [PDF]
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 D. A. McCulloch, E. M. Lutz, M. S. Johnson, D. N. Robertson, C. J. MacKenzie, P. J. Holland, and R. Mitchell ADP-Ribosylation Factor-Dependent Phospholipase D Activation by VPAC Receptors and a PAC1 Receptor Splice Variant Mol. Pharmacol., June 1, 2001; 59(6): 1523 - 1532. [Abstract] [Full Text]
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 S. R. J. Hoare and T. B. Usdin Tuberoinfundibular Peptide (7-39) [TIP(7-39)], a Novel, Selective, High-Affinity Antagonist for the Parathyroid Hormone-1 Receptor with No Detectable Agonist Activity J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 761 - 770. [Abstract] [Full Text]
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W. B. Sneddon, F. Liu, F. A. Gesek, and P. A. Friedman Obligate Mitogen-Activated Protein Kinase Activation in Parathyroid Hormone Stimulation of Calcium Transport But Not Calcium Signaling Endocrinology, November 1, 2000; 141(11): 4185 - 4193. [Abstract] [Full Text] [PDF]
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M. Mannstadt, H. Juppner, and T. J. Gardella Receptors for PTH and PTHrP: their biological importance and functional properties Am J Physiol Renal Physiol, November 1, 1999; 277(5): F665 - F675. [Abstract] [Full Text] [PDF]
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S. R. J. Hoare, T. I. Bonner, and T. B. Usdin Comparison of Rat and Human Parathyroid Hormone 2 (PTH2) Receptor Activation: PTH Is a Low Potency Partial Agonist at the Rat PTH2 Receptor Endocrinology, October 1, 1999; 140(10): 4419 - 4425. [Abstract] [Full Text]
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S. R. J. Hoare, T. J. Gardella, and T. B. Usdin Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor. EFFECT OF RECEPTOR-G-PROTEIN INTERACTION ON THE LIGAND BINDING MECHANISM AND RECEPTOR CONFORMATION J. Biol. Chem., March 9, 2001; 276(11): 7741 - 7753. [Abstract] [Full Text] [PDF]
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[Ca2+]i) in distal convoluted tubule
cells.
