Activation of renal adenylate cyclase by forskolin: Assessment of enzymatic activity in animal models of the secondary hyperparathyroid state
Abstract
The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptormediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.
References (32)
- S.V. Bhat et al.
Tetra. Lett
(1977) - M.P. Dubey et al.
J. Ethnopharmacol
(1981) - F.J. Darfler et al.
J. Biol. Chem
(1982) - K. Seamon et al.
J. Biol. Chem
(1981) - T. Pfeuffer et al.
FEBS Lett
(1982) - D.L. Carnes et al.
Biochim. Biophys. Acta
(1980) - S. Kakuta et al.
Biochim. Biophys. Acta
(1978) - D.F. Fitzpatrick et al.
J. Biol. Chem
(1969) - R.A. Nissenson et al.
J. Biol. Chem
(1979) - O.H. Lowry et al.
J. Biol. Chem
(1951)
Life Sci
Arch. Biochem. Biophys
Life Sci
Arzneim. Forsch./Drug Res
IRCS Med. Sci
Cited by (9)
Calcium Homeostasis in Birds
1989, Vitamins and HormonesThis chapter discusses the system of calcium homeostasis in birds. The importance of Ca2+ in controlling a variety of metabolic functions in the body, ranging from muscle contraction to blood coagulation is reviewed. The Ca2+ participates together with inositol phosphates in the mediation of hormone action in the target cell, including the regulation of secretion of several hormones. Although over 99% of body calcium is contained in the skeleton, the metabolic priority of the organism is the maintenance of a constant concentration of calcium in plasma and extracellular fluids, at the expense of bone calcification and in extreme conditions by the net breakdown of skeletal material. Thus, the relatively insoluble calcium phosphates of bone serve as a calcium reservoir to be utilized during need. This function of bone is particularly prominent in female birds during reproduction. The regulation of Ca2+ concentration in body fluids is achieved through the action of a complex feedback control system which includes several subsystems and regulating hormones.
Forms of adenylate cyclase, activation and/or potentiation by forskolin
1986, Archives of Biochemistry and BiophysicsActivation of different forms of adenylate cyclases (AC) by forskolin and displacement of [14,15-3H]dihydroforskolin binding from membranes by forskolin in the absence or presence of specific stimulatory hormone and β,γ-imidoguanosine 5′-triphosphate (Gpp(NH)p) have been studied. These conditions have been used to generate forskolin dose-response curves of AC activation. A plot of enzyme activation versus apparent forskolin-binding showed a linear and a nonlinear relationship, respectively, in the absence or presence of the other two stimulators. The latter relationship can be fitted by two linear regression lines with a defined intercept, the slopes of which represent two distinct binding-activation (B-A) effects. The B-A effects of forskolin for rat adipocyte and liver membranes in the absence of stimulatory hormone and Gpp(NH)p were 10 and 8 (pmol · min−1) · (pmol)−1, respectively. The B-A effects for the same membranes in the presence of the other two stimulators were 69 (high) and 13 (low) (pmol · min−1) · (pmol)−1 for adipocyte membrane, and 83 (high) and 9 (low) (pmol · min−1) · (pmol)−1 for liver membrane. The ratio of potentiation of forskolin-activated enzyme activity to the unmodified forskolin-stimulated activity (P-A ratio) was determined without the binding data. At 3 μm forskolin, with and without 230 epinephrine and 10 μm Gpp(NH)p, the P-A ratio was 3.7, decreasing to 1.1 with the addition 100 μm forskolin. The line representing a high B-A effect and a resulting high P-A ratio appears to describe the interactions between forskolin and the AC stimulated by epinephrine and Gpp(NH)p. The line of low B-A effect may represent the interaction between forskolin and the basal AC. Two peaks of AC activity were eluted from forskolin-Sepharose column. They have apparent differences in sensitivity to Gpp(NH)p and affinity for forskolin. Based on the results available thus far, with consideration for known limitations of the methodology, a working model has been proposed for forskolin activation of AC.
Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog
1986, Archives of Biochemistry and BiophysicsForskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDPβS. AC in intact membranes of rat or rabbit adipocytes was activated by 100 μm forskolin and further potentiated by 10 μm Gpp(NH)p in combination with either 230 μm epinephrine or 50 mU · ml−1 ACTH. GDP (0–1 mm) or GDPβS (0–500 μm) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 μm forskolin alone. Forskolin at 100 μm stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 ± 4 to 160 ± 10 and from 26 ± 2 to 274 ± 21 pmol(mg · min)−1, respectively, in the absence of GDPβS; forskolin-activated activity decreased from 160 ± 10 to 157 ± 6 and from 274 ± 21 to 238 ± 14 pmol(mg · min)−1 in the presence of 500 μm GDPβS. Forskolin-activated solubilized enzyme was further potentiated by 10 μm Gpp(NH)p from 160 ± 10 to 289 ± 52 and from 274 ± 21 to 702 ± 50 pmol(mg · min)−1. GDPβS at 500 μm inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mm NaCl and 100 μm forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDPβS. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDPβS in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.
A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources
1986, Archives of Biochemistry and BiophysicsWe have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and β,γ-imidoguanosine 5′-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 μm for rat liver, 15 μm for rabbit and rat adipocytes and 17 μm for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 μm. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.
Lack of a direct role for cyclic AMP in parathyrin action on phosphate reabsorption by the kidney
1984, FEBS LettersIsolated chick kidney proximal tubule cells have been used in a study of the mechanism by which PTH inhibits Na+-dependent Pi transport in the kidney. Treatment with PTH inhibits Pi uptake by the cells by 13% and stimulates cyclic AMP production by 77%. Forskolin, a potent activator of adenyl cyclase, brought about an 11-fold stimulation of cyclic AMP production by the cells, but in contrast to PTH, the drug had no effect on Na+-dependent Pi uptake. These results provide evidence that PTH action on phosphate transport is not mediated by cyclic AMP.
Forskolin Editing via Radical Iodo- A nd Hydroalkylation
2021, Synthesis (Germany)