The cAMP system in vasopressin-sensitive nephron segments of the vitamin D-treated rat

The present study was undertaken to investigate the cAMP system in isolated vasopressin (AVP)-sensitive segments of the hypercalcemic rat. Hypercalcemia was produced by supplementation of diet with dihydrotachysterol, achieving a mean serum calcium of 12.6 mg%. Maximal urinary concentration was only 1982 +/- 119 mOsm/kg H2O in pair, watered hypercalcemic rats when compared to 2478 +/- 93 mOsm/kg H2O in controls (N = 7) (P less than 0.01). Vasopressin stimulated adenylate cyclase activity at concentrations of vasopressin between 10(-9) and 10(-7) M was indistinguishable in the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD) of tubules dissected from hypercalcemic rats or normocalcemic rats. Likewise, in situ cAMP accumulation in response to 10(-7) M AVP was not significantly different in either OMCD or IMCD of hypercalcemic or normocalcemic rats at either isotonic or hypertonic media conditions. In contrast, while 10(-7) M AVP significantly (P less than 0.05) increased cAMP accumulation in the medullary ascending limb (MAL) of normocalcemic rats it failed to do so in the MAL of hypercalcemic rats. This failure to accumulate cAMP appears to be due to impairment in AVP-stimulated adenylate cyclase rather than to enhanced phosphodiesterase activity. A similar decrement in glucagon stimulated adenylate cyclase occurred with 10(-6) M glucagon. The results demonstrate that in chronic hypercalcemia the cAMP system in the OMCT and IMCD of the rat is intact, but the MAL demonstrates abnormal AVP responsiveness due to impaired adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indomethacin enhances shuttling of aquaporin-2 despite decreased abundance in rat kidney

The effect of nonsteroidal antiinflammatory drugs on the regulation of aquaporin-2 (AQP2) water channels in the kidney was determined. Male Sprague-Dawley rats were injected with indomethacin (5 mg/kg twice a day intraperitoneally) for 2 d. The control group was injected with vehicle. The expression of AQP2 proteins was determined in the kidney by immunoblotting and immunohistochemistry. The expression of G(salpha) and type VI adenylyl cyclase was determined by immunoblotting. The activity of adenylyl cyclase complexes was determined by stimulated accumulation of cAMP. Immunoblotting revealed that indomethacin markedly decreased the expression of AQP2. Accordingly, however, the ratio of AQP2 expression in the membrane fraction versus that in the cytoplasmic fraction was increased. The urinary excretion of AQP2 proteins also increased. Immunohistochemistry demonstrated almost exclusive apical labeling of AQP2 with scanty cytoplasmic localization along the collecting duct. The expression of G(salpha) and adenylyl cyclase VI proteins was decreased. The generation of cAMP provoked by arginine vasopressin, sodium fluoride, or forskolin was blunted. These results suggest that indomethacin increases the shuttling of AQP2 while it decreases its abundance in the collecting duct.     

Tonic inhibition of adenylate cyclase in cultured hamster Sertoli cells

Sertoli cells cultured from immature hamsters respond to FSH with a dose-related increase in cAMP accumulation. Pertussis toxin acts synergistically with FSH to stimulate cAMP accumulation. This effect of pertussis toxin indicates that Sertoli cell adenylate cyclase is under tonic inhibition due to the activity of the Ni inhibitory transducer. The acetylcholine receptor antagonists atropine or tubocurarine, or the opioid antagonist naltrexone, have no effect on the FSH-induced stimulation of cAMP accumulation, suggesting that neither acetylcholine nor opioids are responsible for the inhibition of Sertoli cell cyclase. While exogenous adenosine is inhibitory, adenosine deaminase augments the ability of FSH to stimulate cAMP accumulation, but not to the level of pertussis toxin. This indicates that the Sertoli cells produce endogenous adenosine that is at least partially responsible for the tonic inhibition of adenylate cyclase. Other possibilities for the tonic inhibition of cyclase include other Sertoli cell products, germ cell products, peritubular cell products or an action of FSH itself through both stimulatory and inhibitory transducers.     

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells

BACKGROUND: Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. METHODS: AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. RESULTS: We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. CONCLUSION: Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.     

Cholinergic inhibition of cAMP accumulation in Sertoli cells cultured from immature hamsters

Acetylcholine inhibits FSH-induced cAMP accumulation in cultured Sertoli cells from immature hamsters. This action of acetylcholine is mimicked by muscarinic cholinergic agonists with a rank order of carbachol greater than acetylcholine greater than arecoline greater than pilocarpine. The carbachol-induced inhibition of stimulated cAMP accumulation is blocked by atropine greater than pirenzepine but not by d-tubocurarine, indicating an apparent muscarinic receptor similar to that found in other peripheral tissues. The fact that pirenzepine is less effective as an inhibitor of the carbachol effect than atropine further defines the muscarinic effect as of the M2 subtype. The ability of carbachol to inhibit FSH-induced cAMP accumulation is blocked by pertussis toxin, which inhibits the action of the Ni inhibitory transducer of adenylate cyclase. These data indicate that cultured Sertoli cells from immature hamsters contain an M2 type muscarinic cholinergic receptor that is negatively coupled to the adenylate cyclase system through the inhibitory Ni transducer.     

Impaired urinary concentrating ability in genetically polyuric mice

Newly recognized strain of mice with hereditary polyuria (PUS mice) was characterized. Polyuria was inherited as a single autosomal-recessive trait. At 15 weeks, PUS mice excreted hypotonic (urine osmolality: PUS;270.8 +/- 15.5 vs. cont.; 3,228.6 +/- 163.6 mosm/kg) polyuria (urine volume: PUS; 25.0 +/- 1.5 vs. cont.; 1.1 +/- 0.1 ml/day). In PUS mice, plasma osmolality was slightly elevated as well as urinary excretion of vasopressin (AVP). Although PUS mice could concentrate urine after 24 h water deprivation, urine osmolality remained low. Blunted response to continuous infusion of dDAVP, a synthetic V2 agonist, was also observed. These in vivo studies indicated renal resistance to AVP contributed to the polyuria in this strain of mice. Microanalysis of isolated tubular segments revealed that AVP-induced cAMP accumulation in cortical collecting ducts (CCD) of PUS mice was significantly lower (60%) with or without a phosphodiesterase inhibitor, IBMX. Vasopressin induced similar cAMP accumulation in medullary ascending limbs of Henle (MAL), and medullary collecting ducts (MCD) between PUS and control mice. In CCDs, PUS mice had low basal adenylate cyclase (AdC) activity and responded less to AVP and forskolin stimulation than control mice. No difference in cyclic AMP phosphodiesterase activity was detected between control and PUS mice. These results indicate that impaired cAMP accumulation due to low AdC activity may be related to the impaired renal concentrating ability observed in this new strain of mice.     

足细胞表达有功能的代谢型谷氨酸受体1和5

目的 研究足细胞是否表达有功能的代谢型谷氨酸受体（mGluR）。 方法 以RT-PCR法检测基因表达,Western印迹、免疫荧光双染色和免疫电镜检测蛋白质表达。酶免疫实验（EIA）、电泳迁移位移实验（EMSA）和Western印迹法检测细胞环腺苷酸（cAMP）的产生和转录因子cAMP反应元件结合蛋白（CREB）的活化。激光共聚焦显微镜观察细胞内游离钙的变化。 结果 足细胞表达mGluR1和5的基因和蛋白质,肾小球中mGluR1的表达和足细胞标志蛋白synaptopodin表达共定位。免疫电镜显示mGluR1位于足细胞体和足突细胞膜下层。mGluR1和5的激动剂3,5-二羟基苯甘氨酸（DHPG）诱导足细胞快速地产生cAMP,转录因子CREB被激活,磷酸化CREB表达增高。这些改变能被mGluR1和5的选择性抑制剂1-氨基茚-1,5-二羧酸（AIDA）以及腺苷酸环化酶抑制剂（SQ22536）抑制,而2-氨基乙氧化联苯-甲硼烷（2APB）则不具有上述抑制作用。DHPG诱导足细胞内游离钙缓慢持续地增加,细胞孵育于不含钙的培养液中、AIDA预处理以及2APB预处理抑制了DHPG诱导的细胞内游离钙离子增加。 结论 足细胞表达有功能的mGluR1和5。     

Arginine vasopressin increases intracellular calcium ion concentration in isolated mouse collecting tubule cells: distinct mechanism of action through V2 receptor, but independent of adenylate cyclase activation.

The effects of arginine vasopressin (AVP*2) and its V2 receptor agonist, 1-deamino-8-D-AVP (dDAVP), on the intracellular calcium ion concentration ([Ca2+]i) in isolated collecting tubular cells of mouse kidney were examined using fluorescent indicator fura-2 and a superfusion system. Both AVP and dDAVP evoked a rapid, transient increase followed by a sustained elevation of [Ca2+]i in CCT, OMCT, and IMCT in a dose-dependent manner. In CCT, the increments in [Ca2+]i by dDAVP were lower than those induced by AVP at all concentrations (10(-10)-10(-6) M) of the agonists tested, while in OMCT and IMCT, the increments were comparable. The initial peak of the rise in [Ca2+]i induced by AVP and dDAVP in these collecting tubule segments was partially attenuated by about 40% and the second sustained elevation was largely abolished in the absence of Ca2+ in the superfusate. Further, the increments [Ca2+]i induced by AVP were not affected by the addition of nicardipine to the superfusate. The increases in [Ca2+]i evoked by AVP and dDAVP were not mimicked by cAMP or forskolin. Moreover, they were not affected by alpha-adrenergic stimulation with epinephrine, in the presence and absence of prazosin, conditions which inhibit AVP-dependent cAMP production. These results indicate that AVP increases [Ca2+]i in CCT, OMCT, and IMCT, probably through V2 receptors, but via a mechanism which is independent of adenylate cyclase activation. In addition, the rise in [Ca2+]i is due to both Ca2+ release from the intracellular stores and increased Ca2+ influx through Ca2+ channels insensitive to nicardipine.     

1,25-dihydroxyvitamin D reduces parathyroid hormone receptor number in ROS 17/2.8 cells and prevents the glucocorticoid-induced increase in these receptors: relationship to adenylate cyclase activation.

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)2D3] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmicphase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. Kd and receptor density were computed from competition of tracer amounts of [125I-Tyr36] hPLP-(1-36) with unlabeled hPLP-(1-36) (0.1-30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)2D3 for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)2D3 (10 nM) completely prevented this receptor increase. Steroid effects required 13-18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC50 of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)2D3 reduction of receptor number in the presence of absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)2D3 (10 nM) treatment. Glucocorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

Renal action of cholera toxin: II. Effects on adenylate cyclase-cyclic AMP system.

The effects of cholera toxin (CT) on the adenylate cyclase-adenosine 3',5'-cyclic monophosphate (cAMP) system(s) in renal cortex were examined using the isolated renal cortical tubules of rat. Unlike parathyroid hormone, catecholamines or prostaglandins, CT had no immediate effects on cAMP production by the tubules or on adenylate cyclase activity. However, after 30 min of incubation at 37 C, cAMP production by the tubules started to rise and reached a plateau between 60 and 90 min. This rise in cAMP production was not abolished by protein synthesis inhibitors (actinomycin D and cycloheximide) nor by the inhibitors of prostaglandin synthesis (acetyl-salicylate and indomethacin). Repeated washings of the tubules exposed to the toxin for five minutes at 0 or 37 C did not abolish the effect of CT to stimulate cAMP production. Assays of adenylate cyclase activity using homogenates prepared from isolated tubules which were incubated for 60 min with CT revealed an increase in the basal adenylate cyclase activity without any change in NaF-sensitive enzyme activity. It is concluded that CT binds to renal tubule cells rapidly, possibly through energy-independent process. CT stimulates adenylate cyclase activity and increases cAMP production by the renal tubule cells after a latent period of 30 min. The stimulatory effects of CT are not due to new protein synthesis or prostaglandin formation.     

Diminished adenylate cyclase activity and aquaporin 2 expression in acute renal failure rats

BACKGROUND: The present study was aimed at investigating the changes of aquaporin 2 (AQP2) expression and its underlying mechanisms in ischemic acute renal failure (ARF). METHODS: ARF was induced by clamping the both renal arteries for 60 minutes in rats. Two or seven days later, AQP2 expression and trafficking were determined in the kidney by Western blot analysis and immunohistochemistry. The activity of adenylate cyclase was also measured. RESULTS: The urinary flow rates in ARF-2 and ARF-7 day were significantly increased in association with decreases of urine osmolality. While there was decreased expression of AQP2 in the cortex, outer medulla, and inner medulla in ARF, it was most pronounced in the outer medulla. The AQP2 expression was reduced in the apical membrane-enriched fraction as well the subapical vesicle-enriched fraction in ARF; however, the degree was greater in the former than in the latter. Immunohistochemical study also showed a markedly decreased expression of AQP2 in the collecting duct in ARF. cAMP generation in response to arginine vasopressin (AVP) in the kidney was attenuated in ARF, most prominently in the outer medulla. cAMP generation in the outer medulla in response to forskolin was not affected, but sodium fluoride was significantly blunted in ARF. CONCLUSIONS: The AVP-stimulated adenylate cyclase activity is impaired in ARF, secondary to a defect at the level of the G protein. The expression of AQP2 was reduced as a consequence, which may in part account for urinary concentration defect in ARF.     

The calcium-ion-sensing receptor

Conclusion The recent cloning, functional, morphologic, and genetic studies have established the CaSR as a vital component of the calcium homeostatic system. The CaSR provides both the sensing mechanism responsible for the regulation of parathyroid hormone secretion from parathyroid cells and the steep relationship between Ca²⁺ ₀ and urinary calcium excretion in the kidney. The renal CaSR appears to provide the crucial “sensing” mechanism in the thick ascending limb and papillary collecting duct for integrating and balancing salt, water, and divalent mineral loss. Direct interactions of extracellular Ca²⁺ with the renal CaSR could explain in large part the disordered water metabolism (ie, nephrogenic diabetes insipidus) observed under pathologic states of hypercalcemia (eg, with primary hyperparathyroidism or associated with certain malignancies). The promise of addition of new calcimimetic agents to our therapeutic arsenal is an exciting prospect. The CaSR story provides an nice example of going from bench to bedside. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology”, Tokyo, May 10, 1998.     

Calcitonin has a vasopressin-like effect on aquaporin-2 trafficking and urinary concentration

The most common cause of hereditary nephrogenic diabetes insipidus is a nonfunctional vasopressin (VP) receptor type 2 (V2R). Calcitonin, another ligand of G-protein-coupled receptors, has a VP-like effect on electrolytes and water reabsorption, suggesting that it may affect AQP2 trafficking. Here, calcitonin increased intracellular cAMP and stimulated the membrane accumulation of AQP2 in LLC-PK1 cells. Pharmacologic inhibition of protein kinase A (PKA) and deficiency of a critical PKA phosphorylation site on AQP2 both prevented calcitonin-induced membrane accumulation of AQP2. Fluorescence assays showed that calcitonin led to a 70% increase in exocytosis and a 20% decrease in endocytosis of AQP2. Immunostaining of rat kidney slices demonstrated that calcitonin induced a significant redistribution of AQP2 to the apical membrane of principal cells in cortical collecting ducts and connecting segments but not in the inner stripe or inner medulla. Calcitonin-treated VP-deficient Brattleboro rats had a reduced urine flow and two-fold higher urine osmolality during the first 12 hours of treatment compared with control groups. Although this VP-like effect of calcitonin diminished over the following 72 hours, the tachyphylaxis was reversible. Taken together, these data show that calcitonin induces cAMP-dependent AQP2 trafficking in cortical collecting and connecting tubules in parallel with an increase in urine concentration. This suggests that calcitonin has a potential therapeutic use in nephrogenic diabetes insipidus.     

Evidence that pulsed electromagnetic fields inhibit coupling of adenylate cyclase by parathyroid hormone in bone cells

To investigate the biochemical effects of pulsed electromagnetic fields (PEMF) on bone in particular and on cell membrane-associated activity in general, we have studied the modification by PEMF of cAMP metabolism in primary calvarial bone cells. We report that PEMF inhibited cAMP accumulation stimulated by bovine PTH(1-34) peptide. After a 1-hr PEMF exposure, the cAMP response to PTH (2-7 min) was decreased in exposed cells to 48-70% (p less than 0.05) of the response of unexposed cells; furthermore, this inhibition disappeared after 10-20 min with PTH. This inhibition occurred at submaximal PTH doses (2.4-7.3 nM) and no effect was observed at maximal PTH doses (24 nM). Thus with PEMF, the dose response curve for PTH became 0.5 log unit less sensitive. PEMF did not affect the cAMP response to cholera toxin and forskolin. However, when submaximal doses of both forskolin (0.5-1.0 microM) and PTH (0.24-2.4 nM) were used, forskolin prevented inhibition of cAMP production by PEMF in the range of fields and stimulus epochs which normally inhibit cAMP production. It is proposed that PEMF inhibits PTH-stimulated coupling of the adenylate cyclase system and that this inhibition does not affect the intrinsic activity of the G-protein and the catalytic subunit.     

Endothelin-1 affects AVP-stimulated cAMP but not ANP-induced cGMP productions in cultured rat IMCD cells

Summary: The present study was undertaken to explore a potential interaction of endothelin-1 (ET-1) on vasopressin (AVP)-dependent cyclic 3',5'-adenosine monophosphate (cAMP) and atrial natriuretic peptide (ANP)-dependent cyclic 3',5'-guanosine monophosphate (cGMP) metabolisms in primary cultured rat inner medullary collecting duct (IMCD) cells. Endothelin-1 did not affect ANP-stimulated cGMP accumulation in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX). Levels of 10⁻¹⁰ to 10⁻⁷ mol/LET-1 showed a dose-dependent inhibition of the AVP-dependent cAMP accumulation in the presence of IBMX and 10⁻⁷ mol/LET-1 inhibited the cAMP generation by 34 ± 5.3% ( n = 5, n = 20, P <0.01). Endothelin-1 did not affect the cAMP generation either by 100 ng/mL cholera toxin or by 10⁻⁴ mol/L forskolin. Endothelin-1 failed to inhibit the cAMP generation in the presence of 100 ng/mL pertussis toxin (PTX). the inhibitory effect of ET-1 was reversed by 10⁻⁸ mol/L staurosporin (SSP), a protein kinase C (PKC) inhibitor. Furthermore, this inhibitory effect of ET-1 was mimicked by 10⁻⁸ mol/L phorbol 12-myristate 13-acetate (PMA), an activator of PKC. A dose of 5 × 10⁻⁶ mol/L indomethacin did not affect this inhibitory effect of ET-1. From these results, we suggest that the effect of ET-1 on cyclic nucleotides metabolism seem to be selective. Endothelin-1 did not affect the cGMP generation by ANP, whereas it inhibited cAMP production by AVP via PTX and SSP sensitive pathway in cultured rat IMCD cells.     

Divergent effects of forskolin on 3′,5′ cyclic adenosine monophosphate production and parathyroid hormone secretion

Summary Forskolin, a diterpene which directly stimulates adenylate cyclase, markedly stimulated cAMP production in intact rat parathyroid glands and dispersed cells from hyperplastic and adenomatous human parathyroid tissues. Stimulation of cAMP production in human parathyroid adenomas occurred as early as 2 min and continued for at least 2 h; furthermore, a dose-response relationship was observed, with a maximal 80-fold cAMP response occurring at 100 μM forskolin. When PTH secretion by rat or human parathyroid tissues was studied at low (0.5 mM) and high (2.5 mM) extracellular Ca²⁺ in either the presence or absence of forskolin, no significant stimulation by forskolin was observed at 15 min, 1 h, and 2 h. When 10 human parathyroid specimens were studied with varying concentrations of forskolin at 1 mM Ca²⁺, 6 failed to show stimulation of PTH secretion and 4 showed modest but detectable increases in PTH that did not appear dose-related. We conclude that (1) at low and high Ca²⁺ levels, marked stimulation of cAMP production by forskolin can occur without a corresponding increase in PTH secretion; (2) inhibition of PTH secretion by high extracellular Ca²⁺ levels continues unchanged despite stimulation of cAMP production by forskolin; and (3) at intermediate Ca²⁺ levels (1.0 mM), PTH secretion is affected either minimally or not at all by forskolin in human hyperparathyroid tissue preparations. The marked stimulation of parathyroid adenylate cyclase by forskolin without concomitant increases in PTH secretion in the majority of tissues suggests that the level of cAMP production is not a primary or sufficient determinant of hormone secretion.