Stimulation of calcium uptake by parathyroid hormone in renal brush-border membrane vesicles. Relationship to membrane phosphorylation.

The effect of parathyroid hormone (PTH) on Ca2+ uptake was studied in brush-border membrane vesicles (BBMV) prepared from the kidneys of dogs administered 4-5 micrograms/kg of bovine PTH 1-84 in vivo. PTH stimulated Ca2+ uptake at 20 s of incubation from control values of 231 +/- 21 to 306 +/- 30 pmol/mg of protein, p less than 0.001. The stimulation of Ca2+ uptake by PTH was not reversed by incubation of the BBMV with the Ca2+ ionophore, despite the fact that Ca2+ uptake was several times greater than the expected uptake at equilibrium, indicating that most of the uptake represented Ca2+ binding to the BBMV. In BBMV from kidneys exposed to PTH, hypotonic lysis or increasing the osmolality of the solution external to the BBMV did not affect Ca2+ uptake. These data also indicated that the largest fraction of Ca2+ uptake in the presence of a chemical potential represented binding of Ca2+ to BBMV. Ca2+ binding was initially to the exterior of the BBMV, then translocated within the membrane and to the interior vesicular face as assessed by chelation of Ca2+ bound to the BBMV by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Incubation of BBMV from kidneys exposed to PTH with gentamicin, which competes with Ca2+ for anionic phospholipid-binding sites, reversed the stimulatory effects of PTH on Ca2+ uptake. Phosphorylation of BBMV and PTH treatment in vivo had similar effects on BBMV phospholipid composition increasing the levels of anionic phospholipids. Phosphorylation of the BBMV also produced gentamicin-inhibitable increases in membrane Ca2+ binding. Phosphorylation of BBMV from kidneys exposed to PTH was inhibited suggesting a higher state of phosphorylation in vivo. The data demonstrate that PTH administered in vivo stimulated Ca2+ binding in BBMV that was gentamicin inhibitable and associated with an increase in the membrane content of anionic phospholipids.

The effect of parathyroid hormone (PTH) on Ca2+ uptake was studied in brush-border membrane vesicles (BBMV) prepared from the kidneys of dogs administered 4-5 rg/kg of bovine PTH 1-84 in vivo. PTH stimulated Ca2+ uptake at 20 s of incubation from control values of 231 2 21 to 306 f 30 pmol/mg of protein, p < 0.001. The stimulation of Ca2+ uptake by PTH was not reversed by incubation of the BBMV with the Caz+ ionophore, despite the fact that Ca2+ uptake was several times greater than the expected uptake at equilibrium, indicating that most of the uptake represented Ca2+ binding to the BBMV. In BBMV from kidneys exposed to PTH, hypotonic lysis or increasing the osmolality of the solution external to the BBMV did not affect Ca2+ uptake. These data also indicated that the largest fraction of Ca2+ uptake in the presence of a chemical potential represented binding of Ca2+ to BBMV. Ca2+ binding was initially to the exterior of the BBMV, then translocated within the membrane and to the interior vesicular face as assessed by chelation of Ca'+ bound to the BBMV by ethylene glycol bis(& aminoethyl ether)-N,N,N',N'-tetraacetic acid.
Incubation of BBMV from kidneys exposed to PTH with gentamicin, which competes with Ca2+ for anionic phospholipid-binding sites, reversed the stimulatory effects of PTH on Ca2+ uptake. Phosphorylation of BBMV and PTH treatment in vivo had similar effects on BBMV phospholipid composition increasing the Ievels of anionic phospholipids. Phosphorylation of the BBMV also produced gentamicin-inhibitable increases in membrane Ca2+ binding. Phosphorylation of BBMV from kidneys exposed to PTH was inhibited suggesting a higher state of phosphorylation in vivo. The data demonstrate that PTH administered in vivo stimulated Ca2+ binding in BBMV that was gentamicin inhibitable and associated with an increase in the membrane content of anionic phospholipids.
The initial step in Ca'+ reabsorption by the renal tubule is Ca2+ translocation from the tubular fluid face of the BBM' to * This work was supported in part by Grant PPG-AM09976 from the National Institute of Arthritis, Metabolism, and Digestive Diseases and Grant-in-Aid 80-721 from the American Heart Association. Parts of this work have been reported in abstract form ((1982) Clin. Res. 30, 451A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Established investigator of the American Heart Association. The abbreviations used are: BBM, brush-border membrane; BBMV, brush-border membrane vesicles; PTH, parathyroid hor-the cystolic side of the membrane. Ca'+ movement across the BBM proceeds along favorable concentration and electrical gradients. This portion of the cell membrane has been reported to be lacking the mechanisms of Ca2+ transport which are associated with Ca2+ efflux from the renal tubular cells. These mechanisms, active transport utilizing energy derived from ATP ((Ca" + Mg2+)-ATPase) and the Na+/Ca'+ exchange mechanism, are functional in the basolateral membranes of renal tubular cells (1,2).
In recent years, preparations of BBMV of renal tubular cells have been utilized to study the solute transport processes of this membrane. Using BBMV with largely a right-side-out orientation, Ca2+ uptake and release have been analyzed (1,3). These studies demonstrate that, in the presence of inwardly directed chemical potentials for Ca2+, uptake represents largely binding of Ca2+ to the vesicular membranes. The bound Ca'+ rapidly distributes within the membrane and to the interior face of the vesicle membrane.
Brush border membrane vesicles have also been utilized to analyze phosphate (Pi) transport. A Na+-dependent co-transport mechanism capable of uphill Pi movement has been characterized in BBMV (4-6). The effects of hormonal stimuli on this Na+-dependent Pi co-transport, including those of PTH, have been investigated (4, 7-9). These studies demonstrated that PTH inhibited Na' dependent Pi co-transport in BBMV by stimulation of cyclic AMP-dependent protein phosphorylation of the BBM. In the course of these studies, cyclic AMP-independent phosphorylation of BBM phospholipids was demonstrated (9). We have subsequently related phosphorylation of anionic BBM phospholipids to an increase in Ca2+ binding in BBMV (3). Since PTH stimulates the metabolism of these phospholipids in isolated renal cortical tubular segments (10) and since PTH is a major regulator of the cellular physiology of calcium (11)(12)(13), including Ca2+ transport (14)(15)(16), the present studies were undertaken to examine the effects of PTH on Ca'+ uptake in BBMV.

Parathyroid
Hormone-stimulated Calcium Uptake 14401 control clearances was obtained. Then, bovine PTH (1-84,3000 units/ mg) was given intravenously, 4-5 pg/kg, body weight. Twenty min later urine was collected for 10 min and the experimental kidney was removed. The renal artery of each kidney was immediately catheterized and perfused with 75-100 ml of ice-cold saline and the kidneys were placed in ice. BBMV were isolated from homogenates of both kidneys by the MgC12 precipitation technique described previously in detail (4,9,17). Isolated BBMV were washed, recentrifuged, and resuspended in 10 mM MgCl,, 10 mM KF, 240 mM mannitol, 5 mM Mes/Tris, pH 6.5 (suspension solution). Membrane preparations were evaluated by electron microscopy and by measuring specific activities of marker enzymes. The results of these studies have been reported previously. PTH did not affect the microscopic appearance of the membranes or the activities of the enzyme markers (4,9,17).
Phosphorylation of BBMV-In those experiments utilizing phosphorylation of BBMV (Figs. 4 and 5), phosphorylation was carried out in hypotonic solutions to cause opening of the vesicles, allowing access of ATP to the vesicular interior as previously reported (3, 9). The solutions used for this purpose were the suspension solutions without mannitol containing 10 p~ ATP. Unless indicated otherwise, incubation of BBMV with 10 p~ ATP was allowed to proceed for 30 s at 30 "C, before addition of solutions used to initiate Ca2+ uptake. Opening and resealing of BBMV by exposure to hypotonic media was demonstrable by release of tritiated glucose accumulated in the presence of sodium chloride gradients and by uptake of sucrose, which is normally excluded, into the intravesicular space (3).
Measurement of Calcium Uptake in BBMV-Ca2+ uptake was measured by a modification of the Millipore filtration technique of Aronson and Sacktor (18) as previously reported (4). Samples of BBMV containing 100-300 pg of membrane protein (10 pl) were incubated in a shaking water bath for 1 min at 30 "C. Then the hypotonic solutions used to induce phosphorylation were added, and incubation was continued for 30 s at 30 "C, before 200 pl of solutions used to initiate Ca2+ uptake were added. In the majority of experiments where phosphorylation of BBMV was not utilized, ATP was deleted from the hypotonic solution, but the hypotonic addition was made so that all BBMV in the present report were handled similarly. The standard solution used to initiate Ca2+ uptake consisted of 48 mM mannitol, 10 mM MgCl,, 10 mM KF, 5 mM Mes/Tris, pH 6.5, and 25 p~ CaC12 with tracer 45CaC12 (2-5 X lo6 cpm/ml). In some instances, Ca2+ concentrations were buffered by addition of EGTA to the solutions. Ca2+ concentration of all solutions was determined by a Ca2+-specific electrode (Orion Research Inc., Cambridge, MA). The uptake of Ca2+ at selected times was terminated by dilution and rapid filtration of the BBMV incubation medium. Solutions used for dilution consisted of ice-cold 100 mM mannitol, 5 mM Mes/Tris, pH 6.5, or 1 mM Mes/Tris, pH 6.5. In some instances, 5 mM EGTA was added to the solutions used to terminate Ca2+ uptake. Filters were dissolved in liquid scintillation vials containing 10 ml of Aquasol (New England Nuclear), and radioactivity was counted in a liquid scintillation spectrometer (model 460 CD, Hewlett-Packard, Downers Grove, IL). Values for nonspecific retention of 45Ca on Millipore filters were subtracted from the values of all membrane samples. All incubations were carried out in triplicate with freshly prepared membrane vesicles.
The results are expressed as mean & S.E.
In experiments in which the uptake of ~-[2-%]glucose was analyzed, 50 p M D-glUCOSe was added to solutions containing 120 mM NaC1,lO mM MgCl,, 10 mM KF, and 5 mM Mes/Tris, pH 6.5. Glucose uptake was stopped by dilution of the incubation medium with 5 ml of ice-cold 150 mM NaCl and rapid filtration.
Analysis of BBM Phospholipids-Analysis of BBM phospholipids was performed by determination of Pi content of phospholipid extracts separated by TLC. Samples of BBMV were added to 3 ml of ice-cold ch1oroform:methanol (1:l) with tetrabutylammonium sulfate (final concentration 10 mM). The volume of each sample was reduced to 1.5 ml under a stream of N, at 22 "C. Then 1 ml of 0.1 N HCl and 1 ml of chloroform were added, and the samples were centrifuged at 1000 X g for 10 min. The aqueous phases were drawn off and discarded. Then, the lipid phases were washed with theoretical upper phase and recentrifuged 3 times. The interface and upper phases were rewashed and this second lipid phase was combined with the washed lipid phases and stored a t -50 "C in vials coated with aquasil (Pierce Chemical Co.). For TLC, the samples were dried under N,, reconstituted in ch1oroform:methanol (1:1), and spotted on TLC plates precoated with Silica Gel 60 (E. Merck, Darmstadt, Germany). TLC plates were developed unidimensionally in several solvent systems designed for separation of specific phospholipids as previously described (3). In some instances a solvent system not previously reported by us, chloroform, methanol, 10% methylamine (6036:10), was used for separation of the phosphoinositides, phosphatidylcholine and phosphatidylethanolamine from other phospholipids. The respective phospholipids were removed from the TLC plates and analyzed for phosphate content using the method of Bartlett (19).
The reported levels of the phosphoinositides, PA and PC, were adjusted for the recovery of known amounts of each phospholipid added to samples of BBMV prior to extraction or to separate tubes without BBMV and carried through the extraction and analysis procedures. Recovery of the added phospholipids varied between 50-86% from experiment to experiment but were very consistent within experiments. Recovery was not different between BBMV from control kidneys and BBMV from kidneys exposed to PTH.
Materials-45CaC12 (14-17 Ci/g of calcium), ~-[2-~H]glucose (18.1 Ci/mmol), and [y3*Pi]ATP (5500 Ci/mmol) were obtained from New England Nuclear. M F -A T P and authentic phospholipid standards were obtained from Sigma. Calcium ionophore (A23187) was obtained from Calbiochem-Behring, and gentamicin sulfate from Bristol, Syracuse, NY. Other chemicals were of the highest purity available from commercial sources. All solutions were filtered through 0.45-pm Millipore filters on the day of the experiment prior to use.
Analysis of the differences in the results between groups was performed using the paired Student's t test except for the data in Figs. 2 and 4 where analysis of variance was utilized. The significance of differences among sample means was assessed using Duncan's multiple range test (20).

RESULTS
The results of the clearance studies performed prior to the removal of the experimental kidney were similar to those reported previously (4,17). Specifically, the administration of bovine P T H 1-84 did not affect plasma calcium, phosphorus, or creatinine during the time of the study. The creatinine clearance was stable during the urine collections following the first nephrectomy, and the excretion of phosphorus (both absolute and fractional) was increased in each case following PTH administration (data not shown).
Effect of PTH on Ca2+ Uptake-Calcium uptake in BBMV isolated from kidneys exposed to PTH was higher compared to BBMV from control kidneys a t both 20 s (estimate of initial rates) and 90 min (steady state) of incubation (Fig. 1). Under the experimental conditions utilized in these experiments, the driving force for Ca'+ uptake was an inwardly BBMV were prepared and suspended as described under "Experimental Procedures" from control kidneys, removed before administration of PTH, or from kidneys removed 30 min after administration of bovine PTH I-84,4-5 pg/kg, body weight. Uptake of Ca2+ at 20 s (estimate of initial rates) and at 90 min (steady state uptake) was determined as described under "Experimental Procedures." P, uptake in BBMV from kidneys exposed to PTH in uiuo was greater than in BBMV from control ( c ) kidneys; p < 0.01 at 20 s and p < 0.05 at 90 min. by guest on March 24, 2020 http://www.jbc.org/ Downloaded from directed chemical potential for Ca2+. In previous studies (1, 3) using similar experimental conditions Caz+ uptake proceeded over time reaching a steady level of uptake between 60-90 min of incubation. Ca2+ uptake also increased as the Ca2+ concentration in the solution external to the BBMV was increased without showing a definite tendency to saturate. In BBMV from PTH-treated kidneys, these characteristics of Ca2+ uptake were not qualitatively altered (data not shown). These results are in agreement with other reports of Ca2+ uptake in renal and intestinal brush border membrane vesicles (1,3,21).
Ca2+ uptake in the experimental setting employed in the above studies may have represented either binding of Ca2+ to the membrane or movement of Ca2+ into the vesicular space in association with other ions. We have previously shown (3) that Ca'+ uptake in BBMV prepared exactly as those from control kidneys reported above represented largely binding to the exterior face of the BBMV followed rapidly by distribution within the membrane and to the interior face of the membrane. Thus, in the present experiments, we also sought to determine the nature of the Ca'+ uptake in BBMV from kidneys exposed to PTH.
An effect of PTH on the volume of the vesicular space might explain the stimulatory effects of PTH. Steady state glucose uptake has been used to estimate the intravesicular volume of BBMV since there is little binding of glucose to the membrane other than its carrier protein (18,22). We have previously reported that PTH does not affect Na+-dependent glucose transport (3,4,17). As shown in Table I, estimated vesicular volumes determined from steady state glucose uptake were not affected by exposure of kidneys to PTH in uiuo. Using these estimates of the vesicular volumes, the expected Ca2+ uptake at equilibrium ranged from 65 to 70 pmol/mg of protein. Ca'+ uptake at 20 s of incubation was 3-4 times greater than predicted equilibrium values, and at steady state, 15-16 times greater (Table I). These observations were tentatively explained by substantial binding of Ca2+ to the membrane, with Ca'+ in the vesicular space approaching only equilibrium values. An alternative possibility was that of Ca2+ transport against a concentration gradient to account for greater than expected values of Ca2+ uptake at steady state. Additional experiments were performed to distinguish between these possibilities.
Effect of the Ca2+ Ionophore (A23187)"The ea2+ ionophore increases the permeability of biologic membranes to Ca'+. When Ca'+ uptake proceeds against a concentration gradient, e.g. ATP-dependent Ca'+ uptake in basolateral membrane vesicles of rat renal tubules, the ionophore causes a prompt dissipation of the Ca'+ uptake above equilibrium values (1).
The addition of the Ca'+ ionophore (lo-& M) to BBMV isolated from control kidneys increased Ca'+ uptake at 20 s of incubation by 38% (Fig. 2). This probably indicated that equilibrium for Ca'+ had not been reached and that increasing permeability of the BBM to Ca2+ allowed greater uptake to 15.8 f 0.7 be stimulated by the chemical potential. The addition of the ionophore to BBMV from kidneys exposed to PTH in vivo did not decrease the stimulation of Ca2+ uptake by PTH but neither did it cause further stimulation. Thus, PTH did not stimulate concentrative accumulation of Ca2+ in the vesicular space of the BBMV. This is despite the fact that uptake of Ca2+ a t 20 s was 3-to 4-fold above the predicted uptake at equilibrium. This suggests that most of the Ca2+ uptake in the presence of P T H represents binding to the membrane. In addition, the stimulatory effects of the ionophore which were observed in BBMV from control kidneys were not additive to those of PTH. This may indicate that PTH besides increasing uptake also increased permeability of the BBMV to Ca2+ in a manner similar to the ionophore. Effects of Hypotonic Lysis and EGTA Treatment-Lysis of BBMV, associated with stopping Ca2+ uptake, would be expected to release unbound Ca2+ accumulated within the vesicular space. As shown in Fig. 3, only small amounts of the Caz+ accumulated by BBMV (either control or experimental) were released by hypotonic lysis. In similar experiments for Na+dependent glucose uptake, release was 75% of uptake (data not shown) similar to previous results (3). The chelation of Ca2+ bound to the exterior face of BBMV (23-25), by addition of EGTA to isotonic solutions used for stopping Ca2+ uptake, decreased Ca2+ uptake by 70-80% at 20 s of incubation. However, after 90 min of incubation, Caz+ chelation by EGTA in isotonic stop solutions decreased Ca2+ uptake by only 37-46%. In comparison, Ca2+ chelation at 90 min of incubation, when EGTA was added to hypotonic lysis stop solutions, resulted in 60-65% reduction in Ca2+ uptake. These results were similar in BBMV from both control and PTH-treated kidneys. Thus, the general sequence of Ca2+ uptake in BBMV appeared to represent binding to the exterior vesicular face where it was accessible for chelation by EGTA in isotonic solution. This was followed by rapid distribution of Ca2+ within the membrane including appearance on its interior face supported by the increased availability of Ca2+ for che- The Ca ionophore was added to solutions used to initiate 45CaC12 uptake.
Parathyroid Hormone-stimulated Calcium Uptake 14403 lation by EGTA in the presence of hypotonic lysis. PTH did not appear to alter the general sequence of binding and distribution within and to the opposite face of the membrane.
Studies were also performed to analyze the effects of decreasing intravesicular volume on the stimulation of Ca2+ uptake by PTH. Intravesicular volume was decreased by increasing the osmolality of the incubation medium in the range from 108-1500 mosm/kg with an impermeant solute (sucrose) (3, 4). Ca2+ uptake at 20 s of incubation in BBMV prepared from kidneys exposed to PTH exhibited a similar lack of sensitivity to increasing external osmolalities as Ca2+ uptake in BBMV from control kidneys (data not shown) (3). These data further indicated that the major portion of Ca2+ uptake in BBMV from kidneys exposed to PTH in vivo represented binding of Ca2+ to the membrane.
Effects of BBM Phosphorylation and Gentamicin-Since the stimulation of Ca2+ uptake in BBMV by PTH appeared to represent mainly an increase in membrane Ca2+ binding, further studies were performed to analyze the nature of the Ca2+ binding sites. In previous studies, binding of Ca2+ to BBMV was increased by membrane phosphorylation in vitro using exposure of BBMV to small concentrations of ATP to produce membrane phosphorylation (3). The effect of membrane phosphorylation on Ca2+ uptake was reproduced in the studies reported here as shown in the left side of Fig. 4. The stimulation of Ca2+ uptake was associated with an increase in the membrane content of anionic phospholipids which bind calcium (3). As shown in Fig. 4, gentamicin, which binds to anionic phospholipids, inhibited the stimulatory effect of phosphorylation of Ca2+ uptake. We previously suggested that this represented competition between Ca2+ and gentamicin for the binding sites produced by phosphorylation. Since PTH produces a stimulation of protein phosphorylation in BBMV (9,26,27) and an increase in the content of anionic phospholipids in renal cortical tubular segments (lo), the effects of ATP-induced phosphorylation on Ca2+ uptake in BBMV from in the BBMV from PTH-treated kidneys was significant, p < 0.01. kidneys exposed to PTH were studied.
Exposure of BBMV prepared from kidneys of dogs treated with PTH to ATP failed to further stimulate Ca2+ uptake (Fig. 4, right). This is compatible with a stimulation of BBM phosphorylation in vivo since this would impair the ability of ATP to stimulate phosphorylation in vitro and thus limit the effect of ATP on Ca2+ uptake. Also, as shown in Fig. 4, the addition of gentamicin to BBMV from kidneys exposed to PTH returned Ca2+ uptake to levels seen in BBMV from control kidneys, suggesting that the stimulatory effects of PTH were similar to ATP in vitro. Since both gentamicin and Ca" bind to negatively charged phospholipids, reversal of the stimulation produced by PTH may have represented competition between Ca2+ and gentamicin for the binding sites produced by PTH treatment. Alternatively, gentamicin may have acted to reverse the effect of PTH by stimulating degradation of the binding sites produced by the PTH.
To analyze if PTH treatment affected the BBMV in terms of phospholipid content, the studies shown in Table I1 were o c c c c P P P P A : P d AiP . .

FIG. 4. Effects of BBMV phosphorylation and gentamicin
on Ca2+ uptake. BBMV were prepared from control kidneys and kidneys exposed to PTH as described under "Experimental Procedures." Phosphorylation of BBMV was as described under "Experimental Procedures." Exposure to 10 p~ ATP was for 30 s at 30 "C prior to addition of solutions used to initiate CaZ+ uptake. Ca2+ uptake was analyzed after 20 s of incubation as described under "Experimental Procedures" except for addition of gentamicin to the solutions used to initiate Ca2+ uptake where indicated. C, BBMV from control kidneys, without additions; C + ATP, control BBMV phosphorylated by addition of 10 FM ATP as described under "Experimental Procedures"; C + G, 100 PM gentamicin added; C + ATP + G, control BBMV phosphorylated and 100 p~ gentamicin added P, BBMV from kidneys exposed to PTH, additions same as for BBMV from control kidneys. *, Ca2+ uptake > than in control, p < 0.05; +, Ca uptake not different from C; ', Ca2+ uptake not different from C.

TABLE I1
Effects of PTH on BBMVphospholipid content BBMV from control and kidneys exposed to PTH were prepared as described under "Experimental Procedures." Phospholipid extraction, separation by TLC, and analysis by phosphate content was as described,under "Experimental Procedures." Values are mean -t S.E., n = 6. Significant differences are shown between respective columns and were determined by paired Student's t test. cortical BBMV. BBMV from kidneys exposed to PTH were phosphorylated as previously described (3) by addition of [Y-:'~P~]ATP (1-2 pCilO.1 mg of BBMV protein) to hypotonic solutions containing 10 p~ ATP. The reactions were stopped by addition of ice-cold ch1oroform:methanol (1:l) with 10 mM tetrabutylammonium sulfate. Following extraction of BBMV lipids and separation by TLC as described under "Experimental Procedures," the TLC plate was exposed to radiographic film (Kodak XRP-1, Eastman) for 48 h. The major phosphorylated products in the lipid extract were PA, TPI, and DPI (3). In control samples (three left-hand lanes a t 30 s, 5 min, and 30 min of incubation, respectively), phosphorylation resulted in similar levels of 32Pi PA, DPI, and T P I at 30 s and 5 min of incubation. The levels of the radiolabeled phospholipids were decreased at 30 min of incubation (third lane from left). In the BBMV treated with gentamicin (three lanes on right), the "Pi-labeled phospholipid levels were greater than in the control BBMV at 30 s (third from right) and 5 min (second from right) and remained higher a t 30 min (far right) of incubation.
performed. They indicate that PTH administration increased the content of PA and of MPI in BBMV prepared from kidneys exposed to PTH in vivo. These anionic phospholipids are known to bind Cap+ and gentamicin avidly (28-32). The increase in the levels of these phospholipids correlated with the stimulation of Cap+ binding by PTH administration and the inhibition of effect of P T H by gentamicin. These data also demonstrate that the previously reported effects of P T H on PA and MPI phospholipid metabolism in renal cortical tubules (10,33,34) are reflected in the BBM, while the effects of PTH on renal tubular polyphosphoinositide content did not reach statistically significant levels in the BBMV.
As shown in Fig. 5 and Table 111, gentamicin added to BBMV prepared from kidneys exposed to PTH caused an increase in :"Pi-labeled phospholipids after exposure to PTH and a tendency for T P I content to increase at the expense of  BBMV from control and kidneys exposed to PTH were prepared in the suspension solution as described under "Experimental Procedures" and incubated in the same solution containing 25 p~ ''CaCI for 90 min. Then 5O-pl aliquots of calcium-equilibrated BBMV (100-200 pg of protein) were distributed into test tubes, and 10 pl of solution containing 5 mM EGTA were added. Calcium loss was stopped by dilution and rapid Millipore filtration. 0, loss in BBMV from control kidneys; 0, loss from BBMV prepared from kidneys exposed to PTH; A, BBMV from control kidneys with 100 p~ gentamicin added at the time of the EGTA addition; A, BBMV from kidneys exposed to PTH with gentamicin addition. Ca2+ loss from BBMV exposed to PTH was more rapid a t 10 and 20 s of incubation ( p < 0.01, n = 6). Calcium remaining in BBMV a t steady state was greater in BBMV from kidneys exposed to PTH than in control kidneys ( p < 0.05), and this effect was reversed by the addition of gentamicin to BBMV from kidneys exposed to PTH (A). 0, m, addition of 10 p~ A23187 to the solutions containing EGTA.
MPI content. The data in Fig. 5 and Table I11 indicated that gentamicin tended to increase the phosphorylation of MPI producing DPI and TPI. This would tend to increase available Ca"-binding sites, not decrease them. This data tends to favor the possibility that the effect of gentamicin on Ca2+ uptake was due to competition for binding sites produced by PTH rather than through stimulation of binding site degradation.
Effects of PTH on Ca2+ Loss from BBMV-To further analyze Ca2+ movement in BBMV from kidneys exposed to PTH, loss of Ca2+ from BBMV pre-equilibrated with Ca2+ was analyzed as previously described (3). BBMV isolated from Parathyroid Hormone-stimulated Calcium Uptake 14405 kidneys exposed to PTH in vivo had significantly higher initial rates of Ca2+ loss than BBMV of control kidneys (Fig.  6). However, at steady state (30 min) slightly greater amounts of ca2+ were retained in BBMV isolated from kidneys exposed to PTH. Since Ca2+ retained by BBMV at steady state far exceeded expected retention of Ca2+ at equilibrium, and since addition of the Ca2+ ionophore had little effect on the Ca2+ retained by the BBMV, most of the Ca2+ retained by BBMV at steady state probably represented Ca2+ bound to the membranes. The addition of gentamicin to BBMV isolated from kidneys exposed to PTH reversed the increase in Ca2+ retained by the BBMV at steady state to levels seen in the BBMV from control kidneys. This again is compatible with an effect of gentamicin competing for anionic binding sites with Ca2+, and the effect of PTH being to increase these binding sites.

DISCUSSION
PTH is an important regulator of renal tubular transport processes, especially for phosphorus and calcium transport. The biochemical mechanisms through which PTH affects the transport of solutes are largely unknown. In the case of Pi, PTH decreases transport in both the proximal and distal renal tubule. A sodium-dependent Pi carrier is located in the BBM, and it is responsible for uphill transport of Pi into the renal tubular cell. We and others have demonstrated that PTH, administered to dogs, inhibits the rate of Na2+-dependent Pi transport in BBMV isolated from kidneys exposed to PTH in vivo (4,(7)(8)(9). The inhibition by PTH of this active step has been shown to be related to biochemical modification of the BBM. We have demonstrated that CAMP-dependent protein phosphorylation of BBMV stimulated by PTH is associated with a decrease in the rate of Na+-dependent Pi transport (9). The suggestion arose, therefore, that stimulation of membrane phosphorylation may be a general mechanism through which PTH affects cellular physiology.
The data presented in this manuscript show that BBMV isolated from kidneys exposed to PTH in vivo exhibit greater rates of Ca2+ uptake than BBMV from control kidneys. This effect was observed in isolated BBMV exposed to a chemical potential for Ca2+ as the driving force for uptake. In this situation, most of Ca2+ uptake represents binding of Ca2+ to the membrane. PTH treatment did not alter the nature of the Caz+ uptake, but it stimulated the early rates of uptake and the steady state level of Ca2+ within the BBMV. The possibility that the effect of PTH on Ca2+ uptake in BBMV was due to phosphorylation of the BBMV was supported by three sets of data. First, if PTH stimulated phosphorylation of the BBM in vivo, then they might be less susceptible to phosphorylation in vitro. In fact, phosphorylation of BBMV from kidneys exposed to PTH in vivo failed to increase Ca2+ uptake while phosphorylation was stimulatory in BBMV from control kidneys. Second, addition of gentamicin, which binds to anionic phospholipids, to BBMV from PTH-treated kidneys decreased Ca2+ uptake to the levels observed in BBMV from the control kidneys. Third, in renal tubular segments, PTH stimulated production of phosphorylated membrane phospholipids, PA, DPI, and MPI (10, 33,34), and the effects on membrane PA were manifested in the BBMV. These data and the effect of PTH on protein phosphorylation (9) indicate that membrane phosphorylation may be a general mechanism by which PTH affects cellular physiology. They also demonstrate that PTH may increase Ca2+ binding to the BBM of proximal renal tubular cells by increasing the membrane content of PA and MPI. The lack of an effect of PTH on the polyphosphoinositides which it has previously been shown to stimulate (10,33,34) may be related to the rapid turnover of these substances and disappearance of the effects of PTH during preparation of the BBM (35,36). Alternatively, the effect of PTH on polyphosphoinositide metabolism, which was observed in renal tubular segments, may be operative at a location within the cell other than the BBM.
The relationship between PTH-stimulated Ca2+ binding in BBMV and transcellular Ca2+ transport is unknown. Ca2+ binding may contribute to transcellular movement of Ca2+ if, after translocation of bound Ca2+ to the interior membrane face, a mechanism is initiated for moving Ca2+ from the membrane to cellular compartments involved in transcellular movement. At the present time, no direct evidence for this potential mechanism in Ca2+ transport is known. However, both phosphorylation of the membrane and an increase in the membrane-bound Ca2+ would be expected to have dramatic effects on the physiologic properties of the membrane. An effect of PTH on the negative charge density and the Ca2+ bound to the BBM may affect the function of ion channels and carrier proteins within the membrane. Direct analysis of the effect of phosphorylation on membrane stability or fluidity will provide insight into this possibility.