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K Daniel

Publications and source records attributed to K Daniel.

36 records · Page 2Linked to original sources
PubMed1987

Phosphorylation of the beta-adrenergic receptor in intact cells: relationship to heterologous and homologous mechanisms of adenylate cyclase desensitization.

We have recently shown that both heterologous and homologous forms of adenylate cyclase desensitization involve phosphorylation of beta-adrenergic receptors. In order to compare these two reactions, we wished to identify a single cell system in which both processes could be studied. Using the frog erythrocyte, which has been previously shown to exhibit cAMP-independent homologous desensitization, we have found that under appropriate conditions cAMP-dependent heterologous desensitization can be elicited. Incubation of intact cells with the membrane-permeable cAMP analogs dibutyryl cAMP or 8-bromo cAMP promotes about a 50% desensitization of isoproterenol- and prostaglandin E1-stimulated adenylate cyclase activity in a time-, temperature-, and dose-dependent fashion. There is also a 20% desensitization in the abilities of guanine nucleotides (GTP and guanyl-5'-yl-imidodiphosphate) and NaF to stimulate adenylate cyclase maximally. In contrast, there is no effect on forskolin- or MnCl2-stimulated enzyme activities. The desensitization response is specific for cAMP as dibutyryl cGMP, 8-bromo cGMP, or 8-bromo AMP produce little or no desensitization. Incubation of the cells with dibutyryl cAMP does not affect the number of cell surface beta-adrenergic receptors. In contrast, incubation with isoproterenol promotes homologous desensitization and sequestration of the receptors. Incubation of 32P-labeled erythrocytes with either dibutyryl cAMP or isoproterenol promotes a stoichiometric threefold increase in the phosphorylation state of the beta-adrenergic receptor which occurs predominantly on serine residues. However, if the cells are coincubated with both dibutyryl cAMP and isoproterenol then the desensitization of isoproterenol-stimulated enzyme activity and phosphorylation of the beta-adrenergic receptor are greater than those observed with either agent alone. These results indicate that heterologous and homologous desensitization of adenylate cyclase-coupled beta-adrenergic receptors are mediated by different biochemical pathways involving phosphorylation of the receptor protein on distinct sites.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes.

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.

Adenylyl Cyclases↗
PubMed1986

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Adenylyl Cyclases↗
PubMed1985

Modulation of pulsatile luteinizing hormone secretion by ovarian steroids in the rat.

In this study, we have examined the effects of estradiol and progesterone on pulsatile luteinizing hormone (LH) secretion in the rat. In the first experiment, regularly cycling rats were ovariectomized on metestrus and a Silastic implant containing estradiol in oil (7.5, 15 or 30 micrograms/ml) or crystalline progesterone (0.7 or 3 cm long) was placed s.c. immediately after surgery. Rats were cannulated 3 days later and frequent blood samples collected (every 10 min for 4 h) the next day. Estradiol treatment alone produced a dose-dependent suppression in mean LH by decreasing LH pulse amplitude without altering interpulse interval. Progesterone treatment alone had no effect, but when the low progesterone dose was combined with a subthreshold estradiol treatment, the combination decreased LH pulse amplitude, but had no effect on the interval between pulses. The combination of high progesterone and low estradiol completely suppressed LH secretion. In the second experiment, rats were bled 7 days postovariectomy to increase the postcastration LH rise and examine higher steroid treatments. Three progesterone treatments (0.7, 1.5 and 3 cm long implants) were combined with the low estradiol treatment; the high estradiol dose (30 micrograms/ml) was also tested. All treatments decreased LH pulse amplitude without altering interpulse interval, although the highest progesterone treatment again completely suppressed LH in most rats so that no statistical analysis could be performed. No firm conclusions can be drawn about the effects of the high steroid treatments that completely suppressed LH; such an effect could reflect either inhibition of pulse frequency or amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Skeletal muscle beta-adrenergic receptors: variations due to fiber type and training.

To determine the relationship between oxidative capacity and characteristics of beta-adrenergic receptors (beta AR) in skeletal muscle, selected biochemical variables were quantitated in particulate preparations from soleus and gastrocnemius muscle from rats subjected to 10 wk of treadmill running and from three control groups: free-fed, sedentary controls; food-restricted, pair-weighted controls; and animals trained by swimming. Beta AR density and isoproterenol-stimulated adenylate cyclase activity were considerably greater in the slow-twitch oxidative soleus muscle than in the mixed fiber type gastrocnemius in animals from each group (P less than 0.005). Succinic dehydrogenase (SDH) activity of gastrocnemius was increased 23-42% (P less than 0.05) in runners over each of the control groups, concommitantly with a 15-27% increase (P less than 0.05) in beta AR density (Bmax for binding of 125I-cyanopindolol). In 24 animals from all four treatment groups, there was a significant correlation between SDH activity and beta AR density (r = 0.68; P less than 0.001). We conclude that BAR density correlates positively with oxidative capacity in skeletal muscle, but further studies are required to determine the physiological importance of these differences.

Adenylyl Cyclases↗
PubMedDate not supplied

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