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Thiazides stimulate calcium absorption in urinary bladder of winter flounder

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Thiazides stimulate calcium absorption in urinary bladder of winter flounder
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  52 Biochimica et Biophysica Acta 897 (1987) 52-56 Elsevier BBA 73188 Thiazides stimulate calcium absorption in urinary bladder of winter flounder Fuad N. Ziyadeh a,b, Ellie Kelepouris a,b and Zalman S. Agus a.b a Renal Electrolyte Section, Department of Medicine, University of Pennsylvania, Philadelphia, PA and h Mount Desert Island Biological Laborato~, Salsbury Cove, ME U.S.A.) (Received 23 June 1986) Key words: Hydrochlorothiazide; Sodium/chloride cotransport; Sodium-calcium exchange; Ouabain; Distal convoluted tubule Thiazides inhibit voltage-independent NaC absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutrai mucosal Na/C co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCI absorption, we studied the effects of hydrochiorothiazide (HCTZ) on unidirectional 4SCa fluxes and intraceilular electrical potential in short-cir- cuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10--4 M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10 -4 M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyper- polarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCI absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule. Introduction The urinary bladder of higher teleosts contrib- utes significantly to salt and water homeostasis in a manner which is complimentary to the function of the kidney [1]. In fact, the urinary bladder of such animals (e.g., Pseudopleuronectes americanus, or winter flounder) is an extension of the mesonephric duct. Thus, unlike the endodermal Abbreviations: HCTZ, hydrochlorothiazide; Hepes, 4-(2-hy- droxyethyl)- 1-piperazineethanesulfonic acid. Correspondence: Fuad N. Ziyadeh, 860 Gates Pavilion, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. srcin of amphibian urinary bladder, the urinary bladder of higher teleosts is anatomically and functionally an extension of the kidney [1,2] and may represent a suitable transport model of some segment of the distal nephron. The flounder urinary bladder is a high-resis- tance epithelium that actively absorbs sodium chloride in a voltage-independent fashion [2,3]. The rates for Na + and CI- net absorption are virtually equal and exhibit interdependence of each ion on the other [2,3]. Moreover, sodium chloride transport is not dependent on the presence of potassium in the mucosal fluid. Recently, Stokes demonstrated that thiazide diuretics inhibit net sodium chloride absorption presumably by block- ing an electroneutral Na/C1 co-transporter located 0005-2736/87/ 03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)  at the mucosal membrane [3]. Thus, the flounder urinary bladder may prove to be a unique model of sodium chloride absorption and may share several functional similarities with the mammalian distal convoluted tubule. Since thiazides stimulate calcium absorption in the early segment of mammalian distal confoluted tubule while simultaneously inhibiting net sodium chloride absorption [4], we examined in the cur- rent study the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and on intracellular electrical potential in the short-cir- cuited flounder urinary bladder to gain some in- sights into the action of thiazides on calcium transport. Methods The methods used are, with slight modifica- tions, those previously described [3,5]. Urinary bladders were dissected from flounders and mounted as flat sheets in modified Ussing cham- bers. Exposed bladders (area=0.80 cm 2) were continuously short-circuited and were bathed on both sides with a solution containing in mM: Na +, 147.5; CI-, 147.5; K +, 2.5; Ca 2+, 1.5; Mg 2+, 1.0; Hepes, 15.0; glucose, 5.0. The mucosal (M) and serosal (S) chambers were continuously bub- bled with 99% O2/1% CO 2 and the pH was ap- proximately 7.5. Verapamil (10/~M) was added to the fluid which bathed the bladders after dissec- tion and before mounting. This was necessary in order to reduce vigorous contraction of bladder smoth muscle [3,5]. After mounting, however, the verapamil was washed off and fresh bathing fluid without verapamil was added to the mucosal and serosal chambers (10 ml each). Whether transient application of verapamil reduces transepithelial calcium fluxes in this tissue remains unde- termined. It has been previously shown, however, that verapamil does not have any effect on mono- valent ion fluxes in this tissue [3]. Transepithelial 45Ca fluxes were measured as previously detailed for frog skin [6]. One hundred /zCi of 45Ca in 100 /xl were added either to the serosal or mucosal solution and the rate of ap- pearance of the isotope was measured on the opposite side. Samples of 50 /~1 each were taken from both chambers every 15 or 30 min, added to 53 scintillation vials, each containing 9.5 ml Aquasol II (New England Nuclear) and 0.5 ml glacial acetic acid, and the radioactivity was counted by liquid scintillation spectrometry. In preliminary studies, it was determined that unidirectional calcium fluxes reach a steady state within 90 min. Thus, we allowed at least 90 rain for isotope equilibration, followed by two or three 30-min control collections. Hydrochlorothiazide (Sigma) was then added to the mucosal bath and three 15-min collections were taken. In all experiments, the short-circuit current (I~c) was measured by continuously calmping the trans- epithelial voltage to 0 mV. To assess the transepi- thelial resistance (Ra-), the transepithelial voltage was clamped at 10 mV for a few seconds every 5-10 min. The resultant deflection in transepi- thelial current was used to calculate R T as previ- ously described [6]. Bladders exhibiting basal R T below 250 12. cm z were discarded. In a separate group of bladders, potential-sens- ing microelectrodes were used to monitor the in- tracellular electrical potential as previously de- scribed [7]. In this set of experiments, bladders were mounted horizontally, mucosal surface up- wards, and were continuously short-circuited. Cells were impaled across the mucosal cell membrane using conventional open-tip micropipettes filled with 0.5 M KCI [7]. The potential difference was measured with reference to the mucosal medium. The outer tip diameter of the micropipette was less than 0.2 /~m and displayed resistances be- tween 60 and 100 MI2 when tested in the bathing solution. All results are expressed as means + S.E. for the group. Data from average values of control periods were compared with those of experimental periods of the same bladders using a paired t test. Comparison between groups of bladders matched by T was done with an unpaired t test. P > 0.05 was considered not significant (n.s.). Results The mucosa-to-serosa calcium flux (JCa~S), i.e., absorptive flux, exhibited an average value of 104 _+ 20 pmol/cm 2 per h (n = 20 bladders) in the basal state. The average R- in these bladders was 724 + 83 2- cm 2. In another group of 12 bladders  54 exhibiting a comparable R T of 592 + 57 I2-cm 2 (P= n.s.), the serosa-to-mucosa calcium flux (JCaSM), i.e. ,secretory flux, was sligthly larger (180 + 40 pmol/cm 2 per h, P = 0.05). Thus, in the absence of an electrochemical gradient for calcium, the flounder bladder tends to express a small degree of net calcium secretion averaging 76 pmol/cm 2 per h. Fig. 1 depicts the effects of mucosal HCTZ (10 -4 M) on JCa MS and R x In each bladder studied, HCTZ increased JCa MS, on average by 46% (from 137 + 30 to 200 + 40 pmol/cm 2 per h, n=9, P<0.005). As previously reported [3], HCTZ also decreased RT, on average by 14% (from 630 + 101 to 541 + 87 Y2. cm 2, P < 0.005). Fig. 2 shows the effects of mucosal HCTZ (10 4 M) on JCa TM and R T in a separate group of 10 bladders. Here, HCTZ had no effect on JCa TM (161 + 40 vs. 160 _+ 50 pmol/cm 2 per h in control period, P=n.s.), while R T was uniformly de- creased, on average by 18% (from 597 + 64 to 491 + 62 12- cm 2, n = 10, P < 0.001) similar to the reduction in R T shown in Fig. 1. Thus, HCTZ, by preferentially stimulating JCa MS, converted the average basal net secretion of calcium (23 pmol/ cm 2 per h) to net absorption (30 pmol/cm 2 per h). In an additional group of three bladders, we examined the effect of pre-treatment with serosal ouabain (10 -4 M) on the response to mucosal HCTZ (Table I). Ouabain was added at the begin- ning of the isotope equilibration period (90 rain). pmoFcm 2 ll I ) 44 400 360 320 28 24 i 200 160 120 80 40 / J/ CONT HCTZ (N:9) COUNT HCTZ 1100 1000 900 800 700 600 500 400 300 200 100 0 RT (D.cm 2) Fig. 1. The effect of mucosal hydrochlorothiazide (HCTZ), 10 4 M, on the absorptive calcium flux (JCa Ms) and on transepithelial resistance (RT) in nine bladders. * P < 0.005 compared to control period (CONT). 48 44 400 360 320 jc ~ 28 (pmol.cni2hr -~) 24 120 80 40 o CONT HCTZ (N=IO) 1300 1200 * 600 ~fJ.cm 2) {so0 400 3O0 200 100 13 CONT HGT Fig. 2. The effect of mucosal hydrochlorothiazide (HCTZ), 10 -4 M, on the secretory calcium flux (JCa TM) and on transepithelial resistance (RT) in 10 bladders. *P<0.001 compared to control period (CONT). JCa Ms and R T were determined in a 60 rain control period, then in a 45 min experimental period beginning with the addition of mucosal HCTZ (10 -4 M). As shown in Table I, pretreat- ment with ouabain prevented the increase in JCa MS as well as the fall in R T induced by HCTZ addition. Fig. 3 depicts the effects of mucosal HCTZ (10 -4 M) on I~c and on the mucosal membrane potential (ffMC). In each of five bladders studied, HCTZ hyperpolarized ~b Mc by an average of 10.7 + 2.5 mV (P<0.02 compared to virtually no change in stable time control values) from -58.4 TABLE I EFFECT OF OUABAIN PRE-TREATMENT ON THE RE- SPONSE TO HYDROCHLOROTHIAZIDE Ouabain (10 -4 M) was added to the serosal bath at the beginning of the isotope equilibration period followed by de- termination of the absorptive calcium flux (JCa Ms) and transepithelial resistance (RT) in the control period (Period I) and following the addition of hydrochlorothiazide (HCTZ), 10 4 M, to the mucosal bath (Period II). The short-circuit current was virtually zero in both periods (n = 3 bladders). JCa Ms (pmol/cm 2 per h) RT (~2.cm 2 ) Period I Period II (ouabain) (ouabain + HCTZ) 84_+ 14 (P=n.s.) 98_+ 6 667+109 (P=n.s.) 647+_94  - 90 - - 80 - - 70 - ~MC_e ° my) ~ 50 - 4.0 0 (N = 5) 6.0 - Y 5.0- I C 3.0 - ~' (pA'crn~) 2.0- .,-~ 1.0- I i 0 CONT HCTZ eONT HCTZ Fig. 3. The effect of mucosal hydrochlorothiazide (HCTZ), 10-4 M, on short-circuit current (lsc) and on the intracellular electrical potential (~b Mc) measured by impaling bladder cells across the mucosal cell membrane. *P < 0.05 compared to control period (CONT). In the open-circuited state, the re- corded transepithelial voltate was mucosa-positive with respect to the serosal bath. ___13 mV in the basal state, to -69.0 + 10 mV after HCTZ addition. Concomitantly, HCTZ re- duced Isc from 3.0± 1.5 to 1.2± 1.5 A/cm 2 (n = 5, P < 0.05). In the urinary bladder of the winter flounder, Isc is totally accounted for by electrogenic potassium secretion [5]. This effect is responsible for the mucosa-positive transepithelial voltage which is recorded in the open-circuited state [5]. The HCTZ-induced reduction of Is~ is similar to that seen in previously reported studies [3,81. Discussion The results of the current study demonstrate that mucosal treatment by HCTZ, in the absence of an electrochemical gradient for calcium, prefer- entially stimulates the absorptive calcium flux in the urinary bladder of the winter flounder without altering the secretory flux. Thus, HCTZ reverses the tendency for basal net calcium secretion into net calcium absorption. Moreover, HCTZ hyper- polarizes the mucosal membrane potential. The effects of HCTZ in decreasing short-circuit cur- rent and transepithelial resistance confirm previ- ously reported studies [3,8]. The stimulation by HCTZ of the absorptive calcium flux is most likely accounted for by an increase in the rate of transport through a cellular rather than a paracellular (shunt) pathway. This is 55 supported by the absence of any increase in the opposite, or secretory, calcium flux with HCTZ administration, despite a fall in transepithelial re- sistance. It is interesting to note that Stokes has concluded that the reduction in transepithelial re- sistance induced by HCTZ could be totally accounted for by a decrease in the cellular rather than the paracellular component of total tissue resistance [8]. In our studies, the abrogation of the effect of HCTZ on transepithelial resistance by ouabain pre-treatment further supports this con- clusion. The mechanism underlying the HCTZ-induced increase in the absorptive calcium flux remains to be elucidated. Theoretically, it may involve stimu- lation of calcium exit from cells to the serosal medium by one or both of two processes: (Ca 2÷ + Mg2÷)-ATPase or Na/Ca exchanger located at the serosal membrane. Direct evidence for the involvement of either process in the flounder urinary bladder is currently lacking. However, the data from the current study are consistent with a role for a serosal Na/Ca exchanger powered by the serosal-to-cell Na ÷ gradient established by the activity of serosally located (Na÷+ K÷)-ATPase. This gradient, when increased, would result in enhanced calcium exit from the cells to the serosal medium. A steeper electrochemical gradient would be expected with hyperpolarization of the in- tracellular electrical potential and/or a decrease in cytosolic Na ÷ activity as might be anticipated when mucosal NaC1 entry is blocked by thiazides. Conversely, the Na ÷ gradient can be greatly re- duced if cell Na ÷ activity is increased and/or if the intracellular electrical potential depolarizes as would be expected with inhibition of (Na ÷ + K ÷)- ATPase by ouabain pre-treatment, resulting in a marked inhibition of calcium exit from the cell via the putative serosal Na/Ca exchanger. Thus, the results of the current study are consistent with a role for a putative serosal Na/Ca exchange pro- cess in mediating the effects of HCTZ. Further direct evidence may be required to confirm this hypothesis. For instance, it may be necessary to show that serosal Na ÷ substitution results in in- hibition of thiazide-stimulated calcium absorption, and that Na/Ca exchange can be demonstrated in serosal-membrane vesicle preparations. It is noteworthy that the magnitude of the  56 unidirectional calcium fluxes reported in this study are relatively small, perhaps suggesting a low in- trinsic permeability to calcium in the flounder urinary bladder. However, these calcium fluxes are of relatively comparable magnitude to those mea- sured in similar high-resistance epithelia, such as the toad urinary bladder [9] or the isolated frog skin epithelium in the basal state [6]. On the other hand, it appears that the net calcium absorption is larger in magnitude in the mammalian distal con- voluted tubule. For instance, in the isolated per- fused rabbit distal convoluted tubule, net calcium reabsorption averaged 0.21 pmol/mm per min [10]. When expressed in terms of tubule surface area, this value appears much larger than flux measurements obtained in the flounder urinary bladder. In summary, this study expands on some of the functional similarities of transport properties ex- hibited by the urinary bladder of the winter flounder and the early segment of mammalian distal convoluted tubule. In both tissues, thiazide diuretics such as hydrochlorothiazide inhibit NaC1 absorption while simultaneously stimulating calcium absorption. Despite the widespread use of thiazides, there is surprisingly very little informa- tion on their mechanism of action in electrolyte transport. The use of the flounder urinary bladder may prove very helpful in elucidating the cellular mechanisms of the action of thiazides on monova- lent and divalent ion transport. The results of the current study are compatible with the hypothesis that the action of hydrochlorothiazide on calcium transport may be mediated by stimulation of serosal Na/Ca exchange. Further studies are re- quired, however, to confirm this postulate. Acknowledgments This work was supported by National Institutes of Health Research Grant R01-AM-33138 and Training Grant T32-AM-07006. F.N.Z. is a recipi- ent of a fellowship from the National Kidney Foundation and from the Measey Foundation. E.K. is a recipient of a Clinician Scientist Award from the American Heart Association. This work was presented in part at the National Meeting of the American Federation for Clinical Research, Washington, DC, 1986, and appeared in Abstract form (Clin. Res. 34 (1986) 703A). References 1 Renfro, J.L. (1975) Am. J. Physiol. 228 (1), 52-61 2 Renfro, J.L. (1978) J. Exp. Zool. 199, 383-390 3 Stokes, J.B. (1984) J. Clin. Inv. 74, 7-16 4 Costanzo, L.S. (1985) Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17), F527-F535 5 Dawson, D.C. and Andrew, D. (1980) Bull. Mt. Desert Isl. Biol. Lab. 20, 89-92 6 Ziyadeh, F.N., Kelepouris, E., Civan, M.M. and Agus, Z.S. (1985) Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18), F713-F722 7 Kelepouris, E., Agus, Z.S. and Civan, M.M. (1985) J. Membrane Biol. 88, 113-121 8 Stokes, J.B., Blackmore, M. and Lee, I.B. (1984) Bull. Mt. Desert Isl. Biol. Lab. 24, 40-41 9 Rosoff, C.J., Baldwin, G.F. and Bentley, P.J. (1983) Am. J. Physiol. 245 (Regul. Integrative Comp. Physiol. 14), R91-R94 10 Shareghi, G.R. and Stoner, L.C. (1978) Am. J. Physiol. 235 (Renal Fluid Electrolyte Physiol. 4), F367-F375
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