The predictive value of first-trimester anthropometric and ultrasonographic adipose tissue measurements in gestational diabetes mellitus.

AIM: Previous studies in pregnant women reported a strong correlation between first-trimester ultrasonographic visceral adipose tissue (VAT) measurements and gestational diabetes mellitus (GDM) during the following weeks. In this study, we aimed to evaluate the clinical utility of ultrasonographic subcutaneous and visceral adipose tissue measurements between 11th and 14th weeks of gestation to predict GDM during the 24th to 28th gestational weeks. MATERIALS AND METHODS: This prospective study included a total of 142 pregnant women. Between 11th and 14th gestational weeks, we performed blinded measurements of intraperitoneal, perirenal, and subcutaneous adipose tissue thicknesses using ultrasonography. Between 24th and 28th gestational weeks, each participant underwent one-step GDM screening, and the measurement data were assessed for prognostic significance. RESULTS: Of the 142 women included, 19 (8.8%) were diagnosed with GDM. The mean intraperitoneal and maximum subcutaneous fat thickness were 51.59 ± 22.49 and 19.79 ± 12.52 mm, respectively for the GDM group and 39.88 ± 13.73 and 13.24 ± 5.70 mm, respectively for the non-GDM group. Although we observed statistically significant differences between the GDM and non-GDM groups in terms of current body mass index (BMI), subcutaneous and intraperitoneal fat thicknesses, and waist and hip circumference values, the logistic regression model showed that only current BMI had a significant association with the increasing GDM frequency. CONCLUSION: In this study investigating the clinical utility of first-trimester ultrasonographic VAT measurements during the 24th to 28th gestational weeks for the diagnosis of GDM, BMI seems to be a more useful predictor than the other anthropometric tools. However, further large-scale studies are needed to confirm these findings.

Osteonecrosis and spontaneous exfoliation of dental implants associated with oral bisphosphonate therapy: a case report.

Bisphosphonates (BPs) have long been used for the treatment of osteoporosis and diseases like bone malignancies, active Paget's disease of bone, severe osteogenesis imperfecta and fibrous dysplasia among others. They bond highly to the bone surface and inhibit bone resorption. As BPs have a long half-life in bone because of their irreversible binding to bone, patients retain their risk profile even after drug cessation. This property also explains the complications wherein the cessation of bone resorption leads to halt in bone turnover. Usually with alendronate the risk of bisphosphonate-related osteonecrosis of jaw (BRONJ) wears off after 12 months. Reporting of the development of BRONJ is commonly associated with the intravenous BPs. Invasive surgical procedure associated with the placement of implants has been shown to be a major reason for the occurrence or initiation of BRONJ in susceptible patients. The prognosis of implants placed in the jaws of patients under or past BP medication is still uncertain. The present case report describes a patient on long-term oral BP therapy with spontaneous exfoliation of implant supported bone due to osteonecrosis.

Association of polymorphisms in the peroxisome proliferator-activated receptor gamma gene and osteoarthritis of the knee.

OBJECTIVES: To study the association between two common polymorphisms in the peroxisome proliferator-activated receptor gamma (PPARgamma) gene and susceptibility to, and severity of, osteoarthritis in a French-Canadian population. METHODS: Genomic DNA was obtained from 172 patients with osteoarthritis and 210 ethnically matched healthy controls. Genotyping for the polymorphisms in the PPARgamma gene (Pro12Ala and C1431T) was carried out using polymerase chain reaction-restriction fragment length polymorphism. The standard Kellgren-Lawrence grading score and the French version of the Western Ontario and McMaster Universities Osteoarthritis Index were used to assess the radiological and functional severity of the disease. Estimated haplotypes were generated using the expectation maximisation algorithm. Genotype and allele frequencies were analysed using the chi2 test. RESULTS: Genotype and allele frequencies for either polymorphism in the PPARgamma gene did not differ significantly between patients with osteoarthritis and controls. Moreover, no significant differences were observed after stratification of patients according to age at disease onset, radiological or functional severity. Similarly, haplotype analysis of both polymorphisms in the PPARgamma gene showed no association of any haplotype with susceptibility to, or severity of, osteoarthritis. CONCLUSION: These findings suggest that the examined polymorphisms in the PPARgamma gene do not contribute to susceptibility to, or severity of, osteoarthritis in the French-Canadian population.

Can altered production of interleukin-1beta, interleukin-6, transforming growth factor-beta and prostaglandin E(2) by isolated human subchondral osteoblasts identify two subgroups of osteoarthritic patients.

OBJECTIVE: To determine the capacity of human subchondral osteoarthritic osteoblasts (Ob) to produce interleukin (IL)-1beta, IL-6, transforming growth factor-beta (TGF-beta) and prostaglandin E(2) (PGE(2)), and determine if a relationship exists between IL-1beta, TGF-beta, PGE(2) and IL-6 production. METHODS: We measured the abundance of IL-1beta, IL-6, TGF-beta and PGE(2) using very sensitive ELISA in conditioned-media of human primary subchondral Ob from normal individuals and osteoarthritic patients. Selective inhibition of IL-6 or IL-6 receptor signaling was performed to determine its effect on PGE(2) production whereas the inhibiton of PGE(2) production was performed to determine its effect on IL-6 production. The expression of bone cell markers and urokinase plasminogen activator (uPA) activity was also determined. RESULTS: Osteoarthritic Ob produced all these factors with greater variability than normal cells. Interestingly, the production of IL-6 and PGE(2) by osteoarthritic Ob separated patients into two subgroups, those whose Ob produced levels comparable to normal (low producers) and those whose Ob produced higher levels (high producers). In those cells classified as high osteoarthritic Ob, PGE(2) and IL-6 levels were increased two- to three-fold and five- to six-fold, respectively, compared with normal. In contrast, while using their IL-6 and PGE(2) production to separate osteoarthritic Ob into low and high producers, we found that IL-1beta levels were similar in normal and all osteoarthritic Ob. Using the same criteria, TGF-beta levels were increased in all osteoarthritic Ob compared with normal. Reducing PGE(2) synthesis by Indomethacin [a cyclo-oxygenase (COX) -1 and -2 inhibitor] reduced IL-6 levels in all osteoarthritic Ob, whereas Naproxen (a more selective COX-2 inhbitor) reduced PGE(2) and IL-6 levels only in the high osteoarthritic group. Conversely, PGE(2) addition to osteoarthritic Ob enhanced IL-6 production in both groups. Moreover, the addition of parathyroid hormone also stimulated IL-6 production to similar normal levels in both osteoarthritic groups. In contrast, using an antibody against IL-6 or IL-6 receptors did not reduce PGE(2) levels in either group. The evaluation of alkaline phosphatase activity, osteocalcin release, collagen type I and uPA activity in osteoarthritic Ob failed to show any differences between these cells regardless to which subgroup they were assigned. CONCLUSIONS: These results indicate that IL-6 and PGE(2) production by subchondral Ob can discriminate two subgroups of osteoarthritic patients that cannot otherwise be separated by their expression of cell markers, and that endogenous PGE(2) levels influence IL-6 synthesis in osteoarthritic Ob.

Endogenous prostaglandin E2 and insulin-like growth factor 1 can modulate the levels of parathyroid hormone receptor in human osteoarthritic osteoblasts.

Subchondral bone sclerosis may be important for the onset and/or progression of cartilage loss/damage in human osteoarthritis (OA). OA osteoblasts are resistant to parathyroid hormone (PTH) stimulation, which could explain bone sclerosis via the inhibition of PTH-dependent catabolism. Here, we investigated the molecular mechanism(s) responsible for reduced PTH-dependent cyclic adenosine monophosphate (cAMP) synthesis in OA subchondral osteoblasts. Although cholera toxin (CTX) increased basal cAMP formation in these cells, it failed to stimulate PTH-dependent cAMP synthesis, whereas pertussis toxin (PTX) did not inhibit basal cAMP, yet diminished PTH-dependent cAMP production. Binding of 125I-PTH indicated lower PTH receptor levels in OA than in normal osteoblasts (-50.5 +/- 9.5%). This could be attributed to either reduced expression of the PTH receptor (PTH-R) or altered recycling of existing pools of receptors. Reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated decreased PTH-R messenger RNA (mRNA) levels in OA cells that were highly variable (ranging from -10% to -60%), a situation that reflects disease severity. Interestingly, OA osteoblasts produced more prostaglandin E2 (PGE2) than normal osteoblasts, and using naproxen, a cyclo-oxygenase inhibitor, increased PTH-dependent cAMP formation to a level similar to normal osteoblasts. Because heterologous desensitization can explain a decrease in PTH binding but cannot account for reduced PTH-R expression, we looked at the possible effect of insulin-like growth factor 1 (IGF-1) on this parameter. Blocking IGF-1 signaling with a neutralizing receptor antibody increased 125I-PTH binding in both normal and OA osteoblasts. Conversely, treatments with IGF-1 receptor (IGF-1R) antibody only slightly increased the levels of PTH-R mRNA whereas the addition of IGF-1 significantly reduced PTH-R mRNA levels (-24.1 +/- 7.1%), yet neither PGE2 nor naproxen modified PTH-R levels. These results suggest that both IGF-1 signaling and PGE2 formation repress PTH-dependent response in OA osteoblasts, a situation that can contribute to abnormal bone remodeling and bone sclerosis in OA.

Carprofen simultaneously reduces progression of morphological changes in cartilage and subchondral bone in experimental dog osteoarthritis.

OBJECTIVE: To examine the effect of a nonsteroidal antiinflammatory drug, carprofen, on the structure and metabolism of cartilage and subchondral bone in the experimental osteoarthritic (OA) canine model. METHODS: Experimental Groups 1 and 2 received a sectioning of the anterior cruciate ligament (ACL) of the right stifle joint, and were administered carprofen (2.2 and 4.4 mg/kg/twice daily/po, respectively) for 8 weeks beginning 4 weeks postsurgery. Group 3 received ACL sectioning and no treatment. Group 4 was composed of unoperated normal dogs. Cartilage macroscopic lesions were assessed, and their histological severity was graded. Specimens of subchondral bones were fixed, decalcified, and stained with hematoxylin/eosin. The level of metalloprotease (MMP) activity in cartilage was measured. Osteoblast cells were prepared from the subchondral bone. The level of synthesis of osteoblast biomarkers (osteocalcin, alkaline phosphatase), as well as urokinase plasminogen activator (uPA) activity and insulin-like growth factor (IGF-1) in the culture medium, was estimated. RESULTS: Carprofen treatment decreased the width of osteophytes (p < 0.01), the size of cartilage lesions, and the histologic severity of cartilage lesions (p < 0.008). There was no difference in the levels of MMP activity in cartilage between OA and carprofen treated groups. In OA dogs, the subchondral bone plate was thinner and was the site of an extensive remodeling process with numerous lacunae. Dogs treated with carprofen showed a marked decrease in the remodeling activity with normal plate thickness, and subchondral bone morphology resembling that of normal dogs. Osteoblasts from untreated OA dogs showed slightly higher alkaline phosphatase activities and osteocalcin release that reverted back to normal upon carprofen treatment. Moreover, uPA activity and IGF-1 levels were increased in OA dogs and were significantly reduced in carprofen treated dogs. CONCLUSION: Under therapeutic conditions, treatment with carprofen could reduce the progression of early structural changes in experimental OA. Carprofen treatment also delays and/or prevents the abnormal metabolism of subchondral osteoblasts in this model. The hypothesis of a possible link between the protective effect of carprofen and its effect on subchondral bone is of interest in the context of therapeutic intervention.

Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in nod mice.

OBJECTIVE: Antibodies directed against general and specific target-organ autoantigens are present in the sera of human patients and animal models with autoimmune disease. The relevance of these autoantibodies to the disease process remains ambiguous in most cases. In autoimmune exocrinopathy (Sjögren's syndrome), autoantibodies to the intracellular nuclear proteins SSA/Ro and SSB/La, as well as the cell surface muscarinic cholinergic receptor (M3) are observed. To evaluate the potential role of these factors in the loss of secretory function of exocrine tissues, a panel of monoclonal and polyclonal antibodies was developed for passive transfer into the NOD animal model. METHODS: Monoclonal antibodies to mouse SSB/La, rat M3 receptor, and a rabbit polyclonal antiparotid secretory protein antibody were obtained for this study. These antibody reagents were subsequently infused into NOD-scid mice. Saliva flow rates were subsequently monitored over a 72-hour period. Submandibular gland lysates were examined by Western blotting for alteration of the distribution of the water channel protein aquaporin (AQP). RESULTS: Evaluation of the secretory response indicated that only antibodies directed toward the extracellular domains of the M3 receptor were capable of mediating the exocrine dysfunction aspect of the clinical pathology of the autoimmune disease. In vitro stimulation with a muscarinic agonist of submandibular gland cells isolated from mice treated with anti-M3 antibody, but not saline or the isotype control, failed to translocate AQP to the plasma membrane. CONCLUSION: These findings define a clear role for the humoral immune response and the targeting of the cell surface M3 signal transduction receptor as primary events in the development of clinical symptoms of autoimmune exocrinopathy. Furthermore, the anti-M3 receptor activity may negatively affect the secretory response through perturbation of normal signal transduction events, leading to translocation of the epithelial cell water channel.

G proteins regulate calcium channels in the luminal membranes of the rabbit nephron.

The filtered calcium (Ca2+) is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by various hormones implies several steps, including binding of these hormones to the basolateral membrane, interaction with G proteins, liberation of messengers, activation of kinases and finally opening of the channels at the opposite pole of the cells. In the present study, we examined whether the Ca2+ entry through the luminal membranes of proximal and distal tubules is also regulated by G proteins, by a membrane-limited process. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPgammas or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPgammas significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles from 0.53 +/- 0.06 to 0.72 +/- 0.06 pmol/microg/10 s (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPgammas increased 0.5 mM Ca2+ uptake in the absence (from 0.57 +/- 0.02 to 0.79 +/- 0.02 pmol/microg/10 s, p < 0.02) and in the presence (from 0.36 +/- 0.03 to 0.55 +/- 0.03 pmol/microg/10 s, p < 0.02) of Na+. The action of GTPgammas, when present, was dose dependent with a half-maximal effect at 20 microM. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPgammas increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.

Abnormal regulation of urokinase plasminogen activator by insulin-like growth factor 1 in human osteoarthritic subchondral osteoblasts.

OBJECTIVE: Subchondral bone sclerosis is a common feature of osteoarthritis (OA), but the mechanisms responsible for this condition remain unresolved. We investigated the role of insulin-like growth factor 1 (IGF-1) and urokinase plasminogen activator (uPA) in human osteoblasts from subchondral bone obtained from the tibial plateaus of OA patients and normal individuals. METHODS: Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients at surgery and from normal individuals at autopsy. Levels of uPA and PA inhibitor 1 (PAI-1) levels were determined under basal conditions and after IGF-1 stimulation in conditioned media from osteoblasts by enzyme-linked immunosorbent assay. The activity of uPA was evaluated by specific substrate hydrolysis and zymography under basal conditions and after plasminogen stimulation, in the presence and absence of added IGF-1. Plasmin activity was also evaluated by specific substrate hydrolysis. RESULTS: Levels of uPA released by OA osteoblasts were significantly higher than normal. Addition of IGF-1 to osteoblasts significantly reduced uPA protein levels only in OA patients (P < 0.05). In contrast, the addition of uPA to osteoblasts did not modify IGF-1 levels in either normal or OA osteoblasts. Basal uPA activity was higher in OA than in normal osteoblasts. Interestingly, IGF-1 enhanced basal uPA activity in OA specimens in a dose-dependent manner. Addition of plasminogen promoted uPA activity in both normal and OA osteoblasts via a positive feedback loop due to plasmin generation, since this activity was inhibited by both PAI-1 and alpha2-antiplasmin. Unexpectedly, incubation with IGF-1 inhibited this positive feedback of plasminogen-dependent uPA activity in OA osteoblasts, but not in normal osteoblasts, in a dose-dependent manner. Hence, normal osteoblasts were relatively insensitive to IGF-1, whereas the same treatment reduced both uPA levels and plasminogen-dependent uPA activity in OA osteoblasts while it increased basal uPA activity in OA osteoblasts. This could not be explained by PAI-1 protein levels, which were similar in normal and OA osteoblasts in the presence and absence of IGF-1. IGF-1 also reduced plasmin activity in OA osteoblasts while it did not modify this activity in normal osteoblasts. CONCLUSION: These results suggest that in OA osteoblasts, the uPA/plasmin system functions normally, yet IGF-1 inhibits the positive feedback of plasmin on uPA activity. This inhibition may contribute to abnormal IGF-1- and uPA-dependent bone remodeling, ultimately leading to abnormal bone sclerosis in OA.

ATP directly enhances calcium channels in the luminal membrane of the distal nephron.

Calcium (Ca(2+)) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca(2+) channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca(2+) uptake by these vesicles loaded with ATP or the carrier. The presence of 100 microM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca(2+) uptake from 0.57 +/- 0.02 to 0.71 +/- 0.02 pmol/microg per 10 sec (P < 0. 01) in the absence of Na(+) and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/microg per 10 sec (P < 0.01) in the presence of 100 mM Na(+). This effect was dose dependent with an EC(50) value of approximately 40 microM. ATP action involved the high-affinity component of Ca(2+) transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPgammas abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca(2+) transport. Inclusion of 20 microM cytochalasin B decreased 0.5 mM Ca(2+) uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/microg per 10 sec (P< 0.01). However, when both 100 microM ATP and 20 microM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca(2+) uptake by the PT luminal membrane. We conclude that the high-affinity Ca(2+) channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca(2+) channels within this membrane.

Osteoblast-like cells from human subchondral osteoarthritic bone demonstrate an altered phenotype in vitro: possible role in subchondral bone sclerosis.

OBJECTIVE: Osteoarthritis (OA) is accompanied by subchondral bone sclerosis. The present study was undertaken to determine whether osteoblast-like cells in patients with OA show an abnormal phenotype that could contribute to this sclerosis. METHODS: Explants and primary in vitro osteoblast-like cell cultures were prepared from subchondral bone specimens from OA patients or from bone removed at autopsy from individuals showing no signs of OA or metabolic bone disease. We measured the abundance and activity of urokinase plasminogen activator (uPA), and the levels of PA inhibitor (PAI-1) and insulin-like growth factor 1 (IGF-1) in conditioned media from both explants and osteoblast-like cells. The expression of osteoblast phenotypic biomarkers was also evaluated. RESULTS: OA explants showed increased levels and activity of uPA, no changes in PAI-1 abundance, and increases in IGF-1 release, as compared with preparations from normal individuals. In vitro primary osteoblast-like cells showed results similar to the ex vivo findings for uPA, PAI-1, and IGF-1. Primary OA osteoblast-like cells also expressed higher alkaline phosphatase activity and osteocalcin release than normal cells, both under basal conditions and with 1,25(OH)2D3 (1,25-dihydroxyvitamin D) stimulation. Conversely, OA osteoblast-like cells showed blunted cAMP synthesis in response to human parathyroid hormone and prostaglandin E2 in contrast to the finding with normal osteoblast-like cells, a result that could not be attributed to altered adenylate cyclase activity. CONCLUSION: Ex vivo and in vitro results indicate similar altered activities of OA osteoblasts as compared with normal cells. This suggests that an altered phenotype of subchondral osteoblasts may be a contributing factor in human OA.

Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron.

In the rabbit, calcitonin has been shown to enhance calcium (Ca2+) reabsorption in the early distal tubule. The aim of the present study was to investigate the mechanism of this action, using isolated luminal and basolateral membranes of distal tubules. The tubule suspensions were preincubated in the presence or absence of 10(-7) M calcitonin. The luminal or basolateral membranes were subsequently purified and 45Ca transport through the vesicles was measured using the rapid filtration technique. Results were compared with those obtained from proximal tubule membranes. In the proximal tubules, calcitonin had no effect on Ca2+ uptake by luminal membranes. In the distal tubules, the presence of Na+ in the incubation medium strongly decreased the uptake of Ca2+ by luminal membranes. Preincubation of distal tubules with calcitonin partially restored this uptake. We previously reported a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. To determine through which messenger(s) calcitonin enhances Ca2+ transport by the distal tubules, we first confirmed that the hormone stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Therefore, we studied the actions of cAMP and phorbol 12-myristate 13 acetate (PMA) on Ca2+ transport by the distal luminal membranes. Incubation of distal tubule suspensions with dibutyryl cAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. We conclude that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms: the opening of low affinity Ca2+ channels in the luminal membrane and the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.

Ca2+ transport by the luminal membrane of the distal nephron: action and interaction of protein kinases A and C.

We previously reported that parathyroid hormone and calcitonin increase Ca2+ uptake by purified distal luminal membranes. This effect is mimicked by high concentrations of cAMP. However, both hormones stimulate adenylate cyclase and phospholipase C. The purpose of the present study was to investigate the role of the phospholipase C pathway in the hormone action, and the interrelationship between the two messengers. Distal tubules from rabbit kidneys were incubated with dibutyryl cAMP (dbcAMP) or PMA, or both, and Ca2+ uptake by purified luminal membranes was measured by the rapid filtration technique. Incubation of the distal tubules with 1 mM dbcAMP significantly increased Ca2+ transport by the luminal membranes. A dose-response curve showed a half-maximal stimulation with 0.82 mM dbcAMP. In contrast, treatment of the tubules with 10 nM, 100 nM or 1 microM PMA did not influence Ca2+ uptake by these membranes. However, the addition of 100 nM PMA to low concentrations of dbcAMP strongly increased this uptake. The presence of cAMP or protein kinase C inhibitors prevented the effects of either a high concentration of dbcAMP alone or a low concentration of dbcAMP combined with 100 nM PMA. Our laboratory has already reported that Ca2+ uptake by the distal luminal membranes displays two-component kinetics. dbcAMP increased the Vmax of the low-affinity component, whereas a combination of the two messengers stimulated the Vmax of both the low- and high-affinity components. From these results, we conclude that: (1) in the distal tubule cells, activation of both protein kinases A and C is necessary for the stimulation of Ca2+ transport by the luminal membrane; (2) the combined effect of protein kinases A and C involves both components of the Ca2+-transport kinetics.