Topical use of phytotherapic cream (Capilen® cream) to prevent radiodermatitis in breast cancer: a prospective historically controlled clinical study.

AIM: The purpose of this study was to evaluate the ability of a topical phytotherapic product (Capilen® cream) to limit acute radiodermitis and delay the use of corticosteroids in patients with breast cancer (BC). METHODS: From January 2012 to August 2012, 30 consecutive patients, undergoing radiotherapy with adjuvant intent, were invited to use Capilen® cream two times daily two weeks before and during radiotherapy. An historical group was used as an external control. Acute skin toxicity was scored weekly according to RTOG/EORTC criteria. Time of occurrence of acute skin toxicity was taken as endpoint. RESULTS: Compliance was good. Overall, no significative statistical difference was observed in rate of acute radiation dermatitis, 46.7% in experimental arm versus 63.3% in the historical control group, although only 3.3% of Capilen® cream treated patients had a G3 acute radiation dermatitis versus 10% of the control group. A delay in the onset of radition dermatitis in patients treated with Capilen® cream (P=0.04) was showed. CONCLUSION: Our findings suggested that Capilen® cream plays a role in reducing acute radiation dermatitis in breast cancer patients treated with adjuvant radiotherapy. Further evidence is needed to confirm these results.

Predictors of mortality in critically ill patients with COVID-19 and diabetes.

The COVID-19 pandemic has challenged the entire world, and patients with diabetes mellitus (DM) have been particularly affected. We aimed to evaluate predictors of mortality during the first 30 days of hospitalization in critically ill patients with COVID-19 and comorbid DM. This prospective study included 110 critically ill patients admitted with COVID-19 infection. Thirty-two (29%) patients had a previous diagnosis of DM. Clinical variables, laboratory tests, and vascular biomarkers, such as VCAM-1, syndecan-1, ICAM-1, angiopoietin-1, and angiopoeitin-2, were evaluated after intensive care unit (ICU) admission. A comparison was made between patients with and without DM. No difference in mortality was observed between the groups (48.7 vs 46.9%, P=0.861). In the multivariate Cox regression analysis, VCAM-1 levels at ICU admission (HR: 1 [1-1.001], P<0.006) were associated with death in patients with DM. Among patients with DM, advanced age (HR 1.063 [1.031-1.096], P<0.001), increased Ang-2/Ang-1 ratio (HR: 4.515 [1.803-11.308] P=0.001), and need for dialysis (HR: 3.489 [1.409-8.642], P=0.007) were independent predictors of death. Higher levels of VCAM-1 in patients with DM was better at predicting death of patients with severe COVID-19 and comorbid DM, and their cut-off values were useful for stratifying patients with a worse prognosis. Vascular biomarkers VCAM-1 and Ang-2/Ang-1 ratio were predictors of death in patients with severe COVID-19 and comorbid DM and those without DM. Additionally, kidney injury was associated with an increased risk of death.

Growth of school children in different urban environments in Argentina.

BACKGROUND: Nutritional transition has been described in various countries, each showing inherent characteristics. Furthermore, different patterns also appear within the same country. AIM: To compare the nutritional status of schoolchildren, of both sexes, living in two Argentine cities with different urban and environment characteristics, from the perspective of nutritional transition. SUBJECTS AND METHODS: The sample comprised 5355 children (6-13 years) living in Puerto Madryn (Chubut) and General Alvear (Mendoza), Argentina. Weight and height were transformed into Z-scores according to NHANES I- II; underweight, stunting and wasting defined by - 2 SD and overweight and obesity calculated according the cut-off proposed by IOTF. Prevalences of nutritional status were estimated. RESULTS: Comparison of the two cities revealed significant χ² values for the indicators of nutritional status analysed. Puerto Madryn had higher prevalences of overweight and obesity. General Alvear exhibited higher stunting and underweight values. CONCLUSIONS: The cities studied are in different stages of nutritional transition. Puerto Madryn is undergoing growing industrialization and urbanization and thus exhibits characteristics typical of an 'obesogenic' environment. General Alvear, a less complex urban centre, where some cultural patterns related to an agrarian way of life appear to have been retained, is situated at a less advanced stage.

Predictors of mortality in critically ill patients with COVID-19 and diabetes

The COVID-19 pandemic has challenged the entire world, and patients with diabetes mellitus (DM) have been particularly affected. We aimed to evaluate predictors of mortality during the first 30 days of hospitalization in critically ill patients with COVID-19 and comorbid DM. This prospective study included 110 critically ill patients admitted with COVID-19 infection. Thirty-two (29%) patients had a previous diagnosis of DM. Clinical variables, laboratory tests, and vascular biomarkers, such as VCAM-1, syndecan-1, ICAM-1, angiopoietin-1, and angiopoeitin-2, were evaluated after intensive care unit (ICU) admission. A comparison was made between patients with and without DM. No difference in mortality was observed between the groups (48.7 vs 46.9%, P=0.861). In the multivariate Cox regression analysis, VCAM-1 levels at ICU admission (HR: 1 [1-1.001], P<0.006) were associated with death in patients with DM. Among patients with DM, advanced age (HR 1.063 [1.031-1.096], P<0.001), increased Ang-2/Ang-1 ratio (HR: 4.515 [1.803-11.308] P=0.001), and need for dialysis (HR: 3.489 [1.409-8.642], P=0.007) were independent predictors of death. Higher levels of VCAM-1 in patients with DM was better at predicting death of patients with severe COVID-19 and comorbid DM, and their cut-off values were useful for stratifying patients with a worse prognosis. Vascular biomarkers VCAM-1 and Ang-2/Ang-1 ratio were predictors of death in patients with severe COVID-19 and comorbid DM and those without DM. Additionally, kidney injury was associated with an increased risk of death.

Renal adenylate cyclase and the interrelationship between parathyroid hormone and vitamin D in the regulation of urinary phosphate and adenosine cyclic 3',5'-monophosphate excretion.

This study examined the role of cyclic AMP in the phosphaturic response to parathyroid hormone in vitamin D-deficient rats. Infusion of purified bovine parathyroid hormone (13.3 mug/h) into control, D-fed, or D-deficient, thyroparathyroidectomized rats produced a sixfold increase in renal phosphate and cyclic AMP excretion in D-fed rats, but only a two- to threefold increase in both parameters in D-deficient animals. Intravenous injection of parathyroid hormone over the dosage range from 1-50 mug/kg resulted in a dose-dependent increase in phosphate and cyclic AMP excretion with both D-fed and D-deficient thyroparathyroidectomized rats. However, the D-deficient rats responded to these injections of parathyroid hormone with a two- to threefold increase in both renal phosphate and cyclic AMP excretion at the highest dose of 50 mug/kg, whereas the D-fed animals' response was 35-fold and 11-fold over control excretion levels of phosphate and cyclic AMP, respectively. To directly examine the role of the renal cortical adenylate cyclase system in the blunted phosphaturic and urinary cyclic AMP responses to parathyroid hormone in D-deficient rats, we prepared a plasma membrane fraction enriched in this enzyme activity from the renal cortex of D-fed and D-deficient thyroparathyroidectomized rats. The renal cortical adenylate cyclase of D-deficient rats showed significantly (P less than 0.001) less activation by parathyroid hormone over the hormone concentration range from 0.3 to 7.0 mug/ml than was observed with the enzyme prepared from D-fed animals. Basal adenylate cyclase activity and the fluoride-stimulated enzyme activity were not altered by the state of D-deficiency. These experiments demonstrate that the blunted phosphaturic response to parathyroid hormone observed in D-deficient rats is associated with the reduced responsiveness of the renal cortical adenylate cyclase to the hormone. Moreover, the defect in the renal membrane adenylate cyclase system appears to be localized at the level of PTH binding to membrane receptors or, alternatively, at the level of transmission of the hormone-receptor binding signal to the catalytic moiety of this membrane enzyme.

Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate.

Intestinal salt and fluid secretion is stimulated by Escherichia coli heat-stable enterotoxins (ST) through activation of a membrane guanylate cyclase found in the intestine. Guanylin is an endogenous intestinal peptide that has structural similarity to the bacterial peptides. Synthetic preparations of guanylin or E. coli ST 5-17 stimulated Cl- secretion in T84 cells cultured on semipermeable membranes as measured by increases in short circuit current (Isc). The guanylin/ST receptors appeared to be on the apical surface of T84 cells, since addition of guanylin to the apical, but not basolateral, reservoir stimulated Isc. Bumetanide added to the basolateral side effectively inhibited the Isc responses of T84 cells to either guanylin or ST 5-17. Guanylin appeared to be about one-tenth as potent as ST in stimulating transepithelial Cl- secretion. Guanylin and E. coli ST 5-17 both caused massive (> 1,000-fold) increases in cGMP levels in T84 cells, but guanylin was less potent than ST. Both peptides fully inhibited the binding of 125I-ST to receptor sites on intact T84 cells. The radioligand binding data obtained with guanylin or ST 5-17 best fit a model predicting two receptors with different affinity for these ligands. The Ki values for guanylin were 19 +/- 5 nM and 1.3 +/- 0.5 microM, whereas the Ki values for ST 5-17 were 78 +/- 38 pM and 4.9 +/- 1.4 nM. We conclude that guanylin stimulated Cl- secretion via the second messenger, cGMP, in T84 human colon cells. At least two guanylin receptors with different affinities for these ligands may exist in the cultured T84 cells. It may be postulated that guanylin is an endogenous hormone that controls intestinal Cl- secretion by a paracrine mechanism via cGMP and that E. coli ST stimulates Cl- secretion by virtue of an opportunistic mechanism through activation of guanylin receptors.

Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP.

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.

Renal parathyroid hormone-dependent adenylate cyclase activity after repletion of vitamin D-deficient rats with vitamin D-2.

Rats fed a diet deficient in both vitamin D and Ca2+ exhibited a greater depression of the renal parathyroid hormone (PTH)-dependent adenylate cyclase than was observed in rats fed diets deficient in either vitamin D or calcium. Total serum Ca2+ was decreased from a control level of 11.2 mg/dl to 8.5 mg/dl in rats fed the diet deficient in calcium alone, and to 5.4 mg/dl in rats fed the diet deficient in vitamin D. Serum calcium was decreased further to 4.3 mg/dl in rats fed the diet deficient in both vitamin D and Ca2+. Serum immuno-reactive PTH was significantly elevated over control levels when rats were fed the test diets; however, there were no significant differences between the elevated levels in the three experimental groups. Repletion of rats deficient in vitamin D only with a single oral dose of 3200 I.U. vitamin D-2 resulted in restoration of serum calcium to normal levels, a return of serum PTH to the control state, and an associated increase in PTH-dependent adenylate cyclase activity to the control level by 72 h. Repletion of rats deficient in both vitamin D and Ca2+ with the same dose of vitamin D-2 raised serum Ca2+ to 7.2 mg/dl by 72 h, but did not cause a reduction in circulating PTH, nor did it result in any significant improvement in the responsiveness of the membrane adenylate cyclase to PTH. These results suggest that elevated PTH is a factor in the down regulation of the PTH-dependent adenylate cyclase, but do not rule out a role for calcium as a regulatory factor.

Cloning of ClC-2 chloride channel from murine duodenum and its presence in CFTR knockout mice.

We report the cloning of a murine ClC-2 chloride channel cDNA from duodenal epithelium by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers and by rapid amplification of cDNA ends (RACE)-PCR. Other than CFTR, this represents the first cloned chloride channel from intact intestine. The ClC-2 cDNA predicts encoding of a 908 amino acid polypeptide with a calculated M(r) of 99,373. The amino acid sequence of the murine ClC-2 chloride channel is over 94% identical to the ClC-2 chloride channel proteins of other species. Of interest is the finding that the ClC-2 mRNA is expressed about the same level in duodena from both CFTR knockout and wild-type mice. This is in keeping with the suggestion that ClC-2 might be a therapeutic target in cystic fibrosis.

Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia.

We have analyzed the expression of Tdt and CD7 in 335 cases of unequivocal acute myeloid leukemia (AML). Tdt was expressed in 80 (25%) of 321 evaluable cases. Twenty-six of 77 (34%) Tdt+ patients assessable for response, entered complete remission (CR) vs 121 of 209 (58%) Tdt- cases (P < 0.001). CD7 was expressed in 102 of 332 (30%) evaluable cases; 37 of 93 assessable (40%) CD7+ patients attained a CR as compared to 114/204 (56%) CD7- (P = 0.013). Duration of survival was significantly shorter for patients with CD7+ or Tdt+ AML (P = 0.006 and 0.001, respectively). In a multivariate analysis, Tdt was found to significantly adverse achievement of CR (P = 0.018), while CD7 affected duration of CR (P = 0.037). Overall the expression of either Tdt or CD7 correlated with a relatively high expression of CD34 (P < 0.001), GP-170 (P = 0.003), lymphoid antigens (LyAg) (P < 0.001), t(9;22) or anomalies of chromosome 5/7 (P < 0.001). Finally, we pooled the patients into four phenotypic classes, according to the presence of Tdt, CD7 or both: [Tdt-CD7-], [Tdt+CD7-], [Tdt-CD7+] and [Tdt+CD7+]. The category [Tdt+CD7+] was characterized by a more unfavorable outcome as suggested by a lower rate of CR (P < 0.001) and a shorter duration of survival as compared to cases [Tdt-CD7-], [Tdt+CD7-] and [Tdt-CD7+] (P = 0.002). This figure is consistent with the frequent convergence in the subset [Tdt+CD7+] of GP-170 positivity (P = 0.003), translocation t(9;22), anomalies of chromosome 5 and/or 7 (P < 0.001) and signs of lineage infidelity (deviant expression of lymphoid antigens) (P < 0.001). We conclude that the expression of Tdt or CD7 is associated with an unfavorable outcome and that the combination of both defines a clinical subset with a poorer prognosis due to the significantly higher association with MDR phenotype, and 'poor prognostic' chromosomal abnormalities.

Structure-activity relationships of parathyroid hormone analogs in the opossum kidney cell line.

Structural alterations in the parathyroid hormone (PTH) molecule produce marked changes in biologic activity. We examined the relative sensitivity of PTH-stimulated cAMP formation and PTH-inhibitable Na+-dependent phosphate transport responses to bovine PTH analogs [bPTH-(1-34), bPTH-(1-84), 8,18-norleucine-34-tyrosinamide bPTH-(1-34), bPTH-(7-34)-amide, 8,18-norleucine-34-tyrosinamide bPTH-(3-34), transaminated bPTH-(1-34)] and the human PTH-related peptide of malignancy (1-34) in cultured opossum kidney cells. The rank order of potency for stimulation of cAMP formation was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) much greater than TAbPTH-(1-34). Nle bPTH-(3-34) and bPTH-(7-34) did not affect cAMP formation in intact cells at concentrations up to 10 microM. The rank order of potency for the inhibition of phosphate transport was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) = TAbPTH-(1-34) greater than nle bPTH-(3-34). TAbPTH-(1-34) was a full agonist and inhibited phosphate transport at concentrations that did not increase cAMP formation, but nle bPTH-(3-34) was a partial agonist in spite of its inability to stimulate cAMP formation. Bovine PTH-(7-34) had no effect on phosphate transport. This study indicates that changes in the PTH molecule produce analogs that apparently discriminate between the cAMP-stimulating activity and phosphate transport-inhibiting activities of the native hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of parathyroid hormone-related peptide supplementation of soy protein formulas in the neonatal pig model.

PTH-related peptide (PTHrP) is found in all milks, including human and pig. To define a role for PTHrP in milk, 2-day-old piglets were randomized to receive soy formula devoid of PTHrP or supplemented with 1 nM synthetic PTHrP(1-86) (n = 8 per group). The number of serum samples with detectable PTHrP by immunoassay (Incstar) and radiometric assay (Nichols) was 9 of 33 and 3 of 13 in PTHrP- and 8 of 27 and 3 of 15 in PTHrP+ formula-fed piglets and 8 of 14 and 7 of 12 in naturally suckling piglets, respectively. Serum and urine concentrations of calcium and magnesium and total and bone alkaline phosphatase were similar in both groups at 3, 6, 10, and 17 days of age. No differences were seen in bone mineral content of the tibia measured by single-photon absorptiometry (BMC 0.22 +/- 0.06 and 0.22 +/- 0.10) or dual x-ray absorption (BMC 1.43 +/- 0.36 and 1.31 +/- 0.78) either in vivo or on excised bone or by measurement of Ca, Mg, or P content or total bone ash (1.26 +/- 0.26 and 1.38 +/- 0.28 mg). Intestinal histology, serum intestinal alkaline phosphatase, and net absorption and retention of Ca, Mg, and P in balances from age 11-17 days were all similar. As in humans, however, a developmental pattern was seen for phosphorus regulation in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences during the development of vitamin D deficiency in the rat: serum parathyroid hormone, calcitonin, calcium, and phosphorus.

Male and female Holtzman rats were raised from weaning on a vitamin D-deficient diet (-D). Control (+D) animals were raised on the same diet and orally given 70 IU vitamin D3 twice each week. Serum Ca levels fell at the same rate in -D males and females. In contrast, serum parathyroid hormone increased more rapidly in males than females, but reached the same maximal level in both sexes. There were no sex differences in serum Ca or parathyroid hormone in the +D group. Serum calcitonin increased with age in all groups. This increase began at an earlier age in females than in males in both +D and -D animals. In both sexes, the increase occurred several weeks earlier in +D than in -D animals, and within each of these groups, males had higher phosphate levels than females. Female rats exhibited greater longevity on the -D diet than their male counterparts. Results of this study document prominent sex differences in the endocrine regulation of Ca homeostasis that were revealed during the development of vitamin D deficiency in the rat.

Parathyroid function and magnesium depletion in the rat.

Hypocalcemia is characteristically observed in magnesium deficiency in a number of animal species. Previous studies demonstrated impaired release of PTH in magnesium-depleted hypocalcemic humans. However, an enigma remains in that, unlike in other animals, hypercalcemia, rather than hypocalcemia, accompanies magnesium deficiency in the rat. Because intact parathyroids are necessary for the development of this hypercalcemia, it has been postulated that magnesium depletion stimulates, rather than impairs, PTH secretion in the rat. In an effort to more directly evaluate this thesis, sequential measurements of circulating immunoreactive PTH (iPTH) were made over a 30-day period in rats maintained on a magnesium-deficient diet and in match-fed controls. In the control rats, serum calcium, magnesium, and iPTH remained relatively constant throughout the study. By contrast, during the first 4 days of a low magnesium diet, serum magnesium decreased to 1.0 mg/dl, serum calcium increased moderately, while serum iPTH increased to a mean level that was twice that in controls. After 5 days, when serum magnesium progressively fell to levels less than 0.6 mg/dl and serum calcium continued to rise, serum iPTH fell to levels significantly lower than the control value. In a second set of experiments, the effect of hypocalcemia on circulating iPTH in magnesium deficiency was evaluated. Circulating iPTH was greatly increased and not significantly different in magnesium-deficient and magnesium-replete animals who were rendered chronically hypocalcemic by diets deficient in either calcium or vitamin D. The results of this study indicate that: 1) in the rat, an increase in PTH secretion occurs early in the genesis of magnesium deficiency in the presence of a modest increase in serum calcium; however, the subsequent further increase in serum calcium counteracts the stimulatory effect of hypomagnesemia on PTH secretion; 2) unlike the human parathyroid gland, the rat parathyroid gland responds appropriately to both hypo- and hypercalcemia in magnesium deficiency; and 3) the hypercalcemia that occurs in the magnesium-deficient rat is not due to increased PTH secretion and must be accounted for by another mechanism.

Serum calcium and parathyroid hormone during the reproductive cycle in normal and vitamin D-deficient rats.

This study was designed to investigate the ability of the vitamin D-deficient (-D) rat to maintain calcium (Ca) homeostasis during pregnancy. Serum Ca and immunoreactive parathyroid hormone (iPTH) were measured before and during pregnancy and after lactation in normal (+D) and -D rats. Serum Ca increased from 9.4 +/- 0.3 to 10.2 +/- 0.2 mg/dl during the first 3 days of pregnancy in +D animals and then declined throughout the remainder of gestation to 9.7 mg/dl at 17 days of gestation. In contrast, serum Ca rose progressively during the first 17 days of pregnancy in -D rats from 5.5 +/- 0.2 to 6.7 +/- 0.4 mg/dl. As expected, the -D rats had markedly elevated serum iPTH, but there were no changes in circulating iPTH during pregnancy in either animal model, even though serum Ca increased significantly during pregnancy in -D animals and Ca decreased in the +D rats. After lactation, serum Ca of -D rats increased 2 mg/dl before returning to prepregnancy levels. These results suggest that there are physiological mechanisms which appear to be independent of vitamin D and are responsible for elevating serum Ca during pregnancy and lactation.

Rat kidney cells in primary culture: hormone-mediated desensitization of the adenosine 3',5'-monophosphate response to parathyroid hormone and calcitonin.

To investigate the direct actions and the possible cellular mechanisms of parathyroid hormone (PTH) and salmon calcitonin (sCT) action in inducing the desensitization of renal cAMP responses to these hormones, we studied kidney cells in primary culture. Renal cells isolated from neonatal rats were cultured in F-12 medium plus 100 microliters/ml calf serum. The cultured kidney cells reached confluent monolayer in 24 h and remained responsive to hormones, including PTH, sCT, vasopressin, and prostaglandin E1. Pretreatment of the cultured cells with PTH (2.5 X 10(-7) M) or sCT (3 X 10(-7) M) resulted in homologous desensitization in the cAMP responses to these hormones. Both PTH- and sCT-mediated desensitization were time and dose dependent. The EC50 for PTH-mediated desensitization was approximately 3.2 X 10(-9) M, and that for sCT was 2.0 X 10(-10) M. Cells that were desensitized to PTH or sCT showed normal responsiveness to cholera toxin, indicating that the catalytic and coupling units of the adenylate cyclase were not modified, suggesting that the locus for desensitization was at the receptor sites. We also found that the renal cell adenylate cyclase, after stimulation by PTH, was markedly different from that observed with sCT. The cAMP response to PTH was short-lived and readily reversible after removal of the hormone from the medium. Exposure to sCT resulted in stable activation of the adenylate cyclase system which was noted for several hours after the removal of sCT, sCT may form a stable complex with the receptors, thus activating the catalytic unit of the adenylate cyclase for a substantial period of time after removal of the hormone. This mechanism may account for the unique pharmacological efficacy of sCT.

Parathyroid hormone (PTH) secretion: stimulation of PTH secretion by a peptide derived from the adenosine diphosphate-ribosylation factor.

Using preparations of dispersed bovine parathyroid cells, we have investigated the effect of a 16-residue synthetic peptide, ARF-16, which corresponds to the N-terminus of the ADP-ribosylation factor, on the secretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrations in the range of 15-25 microM, the ARF peptide stimulated PTH secretion to a greater degree than low extracellular calcium, and at 25 microM was more effective than isoproterenol. The stimulatory effect of ARF was not dependent on the extracellular calcium concentration over the range of 0.5-3 mM. Upon testing other synthetic peptides of similar size we found no effect on PTH secretion, indicating that the ARF-16 effect is specific. In an attempt to define the structural elements of ARF that are required for activity, we tested several analogs of ARF with amino acids deleted from the N- and C-terminus. Deletion of the 2 N-terminal residues yielded a peptide with substantially reduced activity. Further deletions from the N-terminus yielded an inactive peptide. Similarly, a peptide with deletions of 3 residues from the C-terminus was inactive. Thus, the activity of ARF-16 requires both the N- and C-terminal sequence, suggesting that the 16-residue peptide is the minimal sequence required for full activity. Measurements of cAMP concentrations indicate that the stimulatory effect is not mediated via this second messenger. The ARF peptide does not alter intracellular calcium, suggesting that its effect is not mediated by calcium. Although cells incubated with ARF are vigorously stimulated to secrete PTH, this effect is reversible, as demonstrated by washing cells free of ARF, whereupon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion through a low affinity interaction at the cell surface. Other experiments, in which the capacity for ARF stimulation was abolished after a brief exposure of the cells to trypsin, support this conclusion. Characteristics of the ARF stimulatory effect, such as dose dependency and reversibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on extracellular calcium levels and is not mediated via cAMP, we believe that this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.

Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro.

Renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] from 25-hydroxyvitamin D3 (25OHD3) is increased by PTH. The complete mechanism by which PTH modulates renal 25OHD3 metabolism is not known, but there is some evidence that the stimulation of renal cAMP production by PTH may be important. Therefore, we have used forskolin, a direct activator of adenylate cyclase in the intact tissue, to further investigate the role of cAMP in regulating renal 25OHD3 metabolism. The effect of forskolin on renal 25OHD3 metabolism and renal adenylate cyclase activity was measured using isolated renal slices from thyroparathyroidectomized rats previously fed a vitamin D-deficient, low calcium diet. Forskolin added to renal slices in vitro for 4 h increased renal 1,25-(OH)2-D3 production in a concentration-dependent manner. In separate experiments, forskolin was found to increase tissue cAMP in a concentration-dependent manner when added for 5 min. The concentration of forskolin necessary for half-maximal stimulation of adenylate cyclase was 10 microM, and that needed for half-maximal stimulation of 1,25-(OH)2-D3 production was 1 microM. PTH added to renal slices also increased renal 1,25-(OH)2-D3 production, but the effects of PTH and forskolin were not additive. Inclusion of 1,25-(OH)2-D3 in the incubation medium blocked the effect of forskolin on 1,25-(OH)2-D3 production, but it did not block the effect of forskolin on tissue cAMP content. These studies support the concept that forskolin and PTH modulate renal 25OHD3 metabolism though a cAMP-dependent pathway. However, this pathway may be further regulated at sites distal to cAMP production by compounds such as 1,25-(OH)2-D3.

Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: adenosine 3',5'-monophosphate-dependent and -independent mechanisms.

The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.