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Biomedical subjects

L R Forte

Publications and source records attributed to L R Forte.

At least 91 records · Page 5Linked to original sources
PubMed1982

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.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Reduction of brain calcium after consumption of diets deficient in calcium or vitamin D.

Rats fed diets deficient in calcium or vitamin D for 4 weeks displayed hypocalcemia, as indicated by a 50% reduction in serum calcium and a sevenfold elevation of serum parathyroid hormone. These treatments also decreased the calcium content of brain tissue. On a regional basis, this effect was greatest in the brain stem (24% decrease) and least in striatum (10% decrease). Subcellular analysis indicated that the depletion of brain calcium was greatest in the soluble and the microsomal fractions. Infusion of calcium solutions reversed the depletion of brain calcium produced by dietary deficiencies. In control rats, parathyroidectomy or infusion of parathyroid hormone did not alter the calcium content of brain tissue, although these treatments affected the levels of calcium in the serum. In general, these treatments had no effect on the magnesium content of serum or brain tissue. However, vitamin D deficiency did increase the magnesium content of the myelin and synaptosomal fractions. This increase was reversed by parathyroidectomy. These observations demonstrate that long-term hypocalcemia produces distinct changes in the localization of calcium and magnesium in brain tissue. Furthermore, these studies suggest that though brain calcium levels are influenced by serum concentrations, serum changes must be of large magnitude and long duration for brain calcium levels to be affected.

Animals↗
PubMed1981

Regulation of calcium homeostasis in the fetal and neonatal rat.

Circulating levels of calcium (Ca) and immunoreactive parathyroid hormone (IPTH), and the renal cyclic AMP responses to PTH, calcitonin (CT), and vasopressin (VP) were measured in fetal and neonatal rats. Serum Ca increased from a mean value of 9.1 mg/dl on the 19th day of gestation to 10.9 on day 20. Circulating IPTH decreased from 875 pg/ml to 213. Serum Ca declined rapidly after birth to a nadir of 7.6 by 3 h and IPTH increased to 2,006 pg/ml, indicating that fetal and newborn parathyroids are capable of responding appropriately to changes in circulating Ca. Renal responsiveness to hormones was assessed in vitro in the presence of methylisobutylxanthine, a phosphodiesterase inhibitor. The tissue cyclic AMP response to PTH and CT (15- to 18-fold over basal) was greatest at gestational days 18 and 19, progressively declined throughout the remainder of gestation, and remained low during the first 24 h after birth (6- to 7-fold). Renal cyclic AMP response to VP remained consistently low throughout this period. The depressed renal cyclic AMP response to PTH at the time of birth may contribute to the hypocalcemia found in newborn rats.

Adenylyl Cyclases↗
PubMed1981

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.

Aging↗
PubMed1981

Effects of chronic reserpine administration on beta adrenergic receptors, adenylate cyclase and phosphodiesterase of the rat submandibular gland.

Chronic administration of reserpine to rats resulted in a doubling of the density of beta adrenergic receptors in submandibular gland membranes as determined by [3H]dihydroalprenolol binding. The affinity of the labelled drug for the receptor were not altered. There was a concomitant increase in the isoproterenol-stimulated cyclic AMP levels in slices of the gland when incubated in the absence of a phosphodiesterase inhibitor. However, this increase in cyclic AMP accumulation appears to be related to a decrease in phosphodiesterase activity rather than to the increase in the density of beta receptors, since glands from control and reserpinized animals accumulated equal amounts of cyclic AMP when incubated in the presence of a phosphodiesterase inhibitor. In addition, there was no difference between glands from controls and reserpine-treated rats in catecholamine-stimulated adenylate cyclase activity, but the Vmax of the phosphodiesterase was decreased 36% in glands from the treated animals. Similar results were obtained when control and surgically denervated glands were compared. The increase in beta adrenergic receptors appears to be the result of the depletion of norepinephrine due to reserpine administration, whereas the decreased phosphodiesterase activity may result from decreased cyclic AMP levels, secondary to the decrease in norepinephrine.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

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.

Adenylyl Cyclases↗
PubMed1980

Regulation of renal adenylate cyclase by parathyroid hormone.

This study reports the effects of the removal of endogenous PTH by thyroparathyroidectomy (TPTX) on the recovery of the reduced renal cAMP response to parathyroid hormone (PTH) in rats with chronically elevated PTH secondary to diets deficient in either vitamin D or calcium. After TPTX and infusion with a calcium-glucose solution of the vitamin D-deficient rat, calcium and PTH fell from 5.8 mg/dl and 2,509 pg/ml, respectively, to 4.8 mg/dl and 160 pg/ml at 48 h. There was a partial restoration of response to PTH, assessed by assay of renal cortical adenylate cyclase activity from 64% of control activity prior to TPTX to 84% of control activity at 48 h. When rats fed the diet deficient in calcium were TPTX, serum PTH fell rapidly from 2,811 to 200 pg/ml at 5 h with no further change at 21 h, whereas calcium did not change (5.3 mg/dl). PTH-dependent adenylate cyclase activity increased from 59% of control activity prior to TPTX to 87% at 5 h and 100% of control activity at 21 h after TPTX. Each diet produced similar increases in the serum level of immunoreactive PTH, and the rate of disappearance of the circulating hormone after TPTX was also similar for both groups of rats. The data indicate a slow, partial recovery of the enzyme response to PTH after TPTX of the vitamin D-deficient rat over the time period studied, whereas the recovery was rapid and complete in rats fed the diet deficient in calcium.

Adenylyl Cyclases↗
PubMed1978

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat.

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D2, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parthyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the "down-regulation" of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.

Adenylyl Cyclases↗
PubMed1977

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.

Affinity Labels↗
PubMed1976

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.

Adenylyl Cyclases↗
PubMed1976

Impaired release of parathyroid hormone in magnesium deficiency.

Parathyroid hormone release and end-organ responsiveness to parathyroid extract (PTE) were evaluated in a 25-year-old woman with magnesium deficiency associated with hypocalcemia and inappropriately low levels of serum immunoreactive parathyroid hormone (iPTH). End-organ responsiveness to PTE was demonstrated by increases in serum calcium and in urinary phosphorus, cyclic AMP, and hydroxyproline. When the serum calcium was increased from a baseline of 6.9 mg/100 ml to levels of 8.0 mg/100 ml and higher by calcium infusion, the serum iPTH decreased from the low normal range to below the limits of detectability. The intravenous administration of 3 mg/kg of body weight of magnesium led to an abrupt and striking increase in circulating iPTH with a 2-fold increase in one minute, a 6-fold increase in two minutes, and an 8-fold increase in five minutes. The very rapid increase in serum iPTH produced by magnesium infusion in this study suggests an effect of magnesium on hormone secretion rather than an effect on hormone synthesis. The evidence provided by this investigation indicates that the release of parathyroid hormone is impaired in magnesium deficiency and that the level of circulating calcium required for the suppression of parathyroid hormone secretion is lower than that in normal subjects.

Adult↗
PubMed1975

Interaction of parathyroid hormone with membranes of kidney cortex: degradation of the hormone and activation of adenylate cyclase.

Plasma membranes prepared from rat renal cortex contain both a parathyroid hormone-sensitive adenylate cyclase and a potent proteolytic activity which degrades the hormone into peptide fragments. The degree and pattern of degradation was determined by subjecting incubation mixtures to gel filtration and ion exchange chromatography. Estimation of the degree of degradation by acid precipitation of the intact hormone was inadequate since metabolism of the hormone apparently generated acid-insoluble fragments. When parathyroid hormone was incubated with membrane fraction, the capacity of its stimulatory effect on adenylate cyclase decreased steadily. This decrease of PTH activitiy could be closely related to the degradation of intact hormone by the same membrane preparation. The adenylate cyclase and degradative activity appeared to exist in similar membrane structures since they could not be separated by centrifugation through sucrose density gradients. The degradation of the hormone could not be inhibited by Trasylol and pancreatic or soybean trypsin inhibitors and was only slightly inhibited by ribonuclease and benzamidine. Histone (1 mg per ml), on the other hand, was able to decrease the degradation of the hormone and prevent the loss of its activity. Radioimmunoassay of the incubation mixtures showed that the rapid degradation of both amino- and carboxy-terminal regions of the hormone was prevented by histone. The oxidized, inactive hormone was also degraded to the same extent by the renal cortical membrane. Furthermore, the degradative activity was also found in plasma membrane preparations of renal medulla and liver. This lack of hormone and tissue specificity suggests that similar degradative activity exists in all tissues and that caution should be exercised in estimating hormonal potency based on activation of adenylate cyclase.

Adenylyl Cyclases↗