Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.     

Role of increase in intracellular calcium in PTH-induced homologous desensitization in UMR-106 cells.

Effects of increase in intracellular calcium on PTH-induced homologous desensitization were investigated using calcium ionophores. Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with calcium ionophores (A23187 or ionomycin) for 6h resulted in approximately 50% decrease of PTH-stimulated cAMP production. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was significantly decreased in 10(-6) M calcium ionophore-pretreated (for 6h) cells without affecting the dissociation constant (Kd) for PTH. Minimal effective treatment period was 2h and similar inhibitory effect was observed in 12h-treated cells. These data suggest that increase in intracellular calcium might also act on PTH receptor in the similar manner as protein kinase C activation to induce desensitization.     

Differential effect of TPA on PGE2 and cicaprost-induced cAMP synthesis in UMR-106 cells.

PGE2 and prostacyclin each enhance cAMP synthesis in the osteoblast-like cell line UMR-106. The amount of cAMP induced by PGE2 was 5-7-fold greater than the amount induced by cicaprost or iloprost, stable prostacyclin analogues. Both PGE2 and the two prostacyclin analogues enhanced cAMP synthesis with similar time dependence. The EC50 values of PGE2 and cicaprost were 3 X 10(-6) and 5 x 10(-8) M, respectively. Short-term incubation of the cells with 12-o-tetradecanoylphorbol 13-acetate (TPA) markedly reduced the PGE2-induced cAMP synthesis. In contrast, cells that were incubated with the same concentrations of TPA in the presence of cicaprost or iloprost showed a 1.6-fold increase in cAMP formation. The marked disparity between the cAMP response to cicaprost and PGE2 in the presence of TPA suggests that the two prostanoids induce cAMP synthesis in the UMR-106 cells by interaction with different receptors. These observations support the idea that the osteoblastic UMR-106 cells may express specific prostacyclin receptors and suggest that prostacyclin may have a unique role in osteoblasts.     

Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.     

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.     

Simultaneous recording of cell volume changes and intracellular pH or Ca2+ concentration in single osteosarcoma cells UMR-106-01.

We present a new technique for the simultaneous measurement of cell volume changes and intracellular ionic activities in single cells. The technique uses measurement of changes in the concentration of intracellularly trapped fluorescent dyes to report relative cell volume. By using pH- or Ca(2+)-sensitive dyes and recording at the ion-sensitive and -insensitive (isosbestic) wavelengths, the method can measure both cell volume changes and intracellular ionic activities. The technique was used to study the mechanisms of regulatory volume decrease (RVD) in the osteosarcoma cell line UMR-106-01 grown on cover slips. Swelling cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hypotonic medium was followed by stable cytosolic acidification and a decrease in cell volume back toward normal. The recovery of cell volume could be blocked by depolarization, treatment with ouabain, or depletion of cell Cl-. These suggest the conductive efflux of K+ and Cl- during RVD. The cytosolic acidification that accompanied cell swelling was not blocked by amiloride, bafilomycin A, or removal of Cl- and could not be reproduced by depletion of cellular ATP. These findings exclude Na+/H+ and Cl-/HCO-3 exchange, intracellularly generated acid, or increased metabolism, respectively, as the cause of the acidification. The cell swelling-induced acidification was inhibited by depolarization, suggesting the involvement of an electrogenic pathway. The acidification, as well as RVD, was inhibited by short incubation with deoxyglucose, and these effects could not be reversed by valinomycin. Thus, the anionic pathway(s) participating in RVD and the acidification are sensitive to the cellular level of ATP. Together, these studies indicate that RVD in UMR-106-01 cells in HEPES-buffered medium is mediated by the conductive efflux of K+, Cl-, and OH-.     

Low-intensity pulsed ultrasound stimulates a bone-forming response in UMR-106 cells

Low-intensity (<100 mW/cm(2)) pulsed ultrasound (US) is an established therapy for fracture repair. In both animal and human trials, such US has been shown to facilitate fresh fracture repair and initiate healing in fractures with repair defects. However, the mechanism by which US achieves these outcomes is not clear. One possible mechanism is the direct stimulation of bone formation. To investigate this hypothesis, the current study investigated the mRNA response of isolated bone-forming cells (UMR-106 cells) to a single 20-min dose of low-intensity pulsed US. Using a novel US-cell coupling method, US was found to stimulate expression of the immediate-early response genes c-fos and COX-2 and elevate mRNA levels for the bone matrix proteins ALP and OC. These findings suggest that low-intensity pulsed US has a direct effect on bone formation. This may contribute to the beneficial effect of low-intensity pulsed US on fracture repair.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.     

Calcium dependence of hormone-stimulated cAMP accumulation in intact glial tumor cells.

The Ca2+ content of glial tumor (C6) cells was reduced approximately 5-fold by repeated treatment with media containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) without loss of cellular viability. The ability of the cells to accumulate cAMP in response to beta-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response. The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of cAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor. Micromolar free Ca2+ concentration in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2+-depeleted cells. The effect of Ca2+ on cAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of cAMP accumulation; with Ca2+-depleted cells cAMP was accumulated more slowly and the subsequent decline in cAMP content was also reduced. Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the cAMP accumulation when added prior to the cation. The effect of Ca2+ on cAMP accumulation was reduced more extensively by pretreatment of cells at 45 degrees C under Ca2+-depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of cAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.     

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Down-regulation of adenylate cyclase-coupled response to native bovine parathyroid hormone and fragments in the osteoblast-like cell line UMR-106.

1. Parathyroid hormone-induced down-regulation was studied in the osteosarcoma cell line UMR-106. 2. A maximal priming does of bPTH (1-84) down-regulated PTH-responsiveness to 40% of its initial value; bPTH (1-41) was less effective than bPTH (1-84), whereas bPTH (42-84) had no effect, alone or in combination with bPTH (1-41). 3. A tentative model for the function of different domains of parathyroid hormone in down-regulation is suggested.     

Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.     

Dissociation between release and gene expression of gonadotropin alpha-subunit in gonadotropin-releasing hormone-stimulated alpha T3-1 cell line.

The alpha T3-1 cell line which was derived by targeted tumorigenesis in transgenic mice [Windle et al. (1990) Mol. Endocrinol. 4, 597-603] possesses high-affinity binding sites for GnRH analogs coupled to enhanced phosphoinositide turnover and phospholipase D activity. Incubation of alpha T3-1 cells with [D-Trp6]-GnRH analog (GnRH-A) resulted in a rapid increase in gonadotropin alpha-subunit mRNA levels which was detected already at 30 min of incubation (0.1 nM GnRH-A, 3-fold, p < 0.01). The effect diminished with time to reach basal levels at about 12 h of incubation, with a secondary rise in alpha mRNA levels between 12 and 24 h of incubation. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 ng/mL) or the Ca2+ ionophore ionomycin (1 microM) to alpha T3-1 cells also resulted in a rapid increase in alpha-subunit mRNA levels. Surprisingly, GnRH-induced alpha-subunit release was detected only after a lag of 4 h of incubation. Thus, dissociation between exocytosis and gene expression can be demonstrated in GnRH-stimulated alpha T3-1 cell line.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR-106 osteoblastic cells

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-alpha (PKCalpha) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1-34, PTH 3-34, PTHrP and PTH 1-31 stimulated PKCalpha translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1-34- or PTH 3-34-stimulated PKCalpha translocation. The phosphoinositol-phospholipase C (PI-PLC) antagonist U73122 slightly decreased PTH 1-34-stimulated PKCalpha translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1-34- and PTH 3-34-stimulated PKCalpha translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-alpha translocation and IL-6 promoter activation in osteoblastic cells.     

Calcium-activated calcium permeability in parathyroid cells

The Ca2+ receptor mechanism of the parathyroid cell was studied using La3+ as a probe. La3+ was found to bind to the cell surface without further penetration. Measurements of 45Ca fluxes and the cytoplasmic Ca2+ concentration (Ca2+i) revealed a stimulatory component in the action of La3+ on Ca2+ permeability resulting in a rise in Ca2+i. These effects mimicked those obtained when raising the extracellular Ca2+ concentration from 0.5 to 3.0 mM, but the actions of La3+ and Ca2+ were not additive. The results suggest the existence of a novel Ca2+ permeability physiologically activated by binding of Ca2+ to an external receptor.     

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

The effects of parathyroid hormone (PTH) on cytoplasmic free CA²⁺ (Ca _i ²⁺ ) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01.In fura-2 loaded adherent single cells bPTH 1-34 (10 nM–1M) induced a rapid transient increase in Ca _i ²⁺ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 M) induced a transient increase in Ca _i ²⁺ in 20% of the experiments. The transient increase in Ca _i ²⁺ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Ca _i ²⁺ was derived from intracellular stores.A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Ca _i ²⁺ .These data show that PTH causes a release of Ca²⁺ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Ca _i ²⁺ in osteoblasts.     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.