Gonadal LH Receptors: Resolution from Adenylate Cyclase and Transfer to Heterologous Cells

Dufau, Maria L.; Hayashi, Kozaburo; Sala, G; Báukal, Albert J.; Catt, Kevin J.

doi:10.1007/978-1-4684-3474-3_4

Cited by 3 publications

(3 citation statements)

References 6 publications

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“…In this sense, this paper shows that such cascade system is in fact reassembled in electrofusion-generated hybrids. The same evidence was presented in granulosa cells [27] and by transfer of functional ovarian luteinizing hormone receptors to adrenal fasciculata cells [28]. We have previously demonstrated that the information to generate a signal for a specific function in rat Leydig cells resides in the receptor and not in the hormone [29].…”

Section: Resultssupporting

confidence: 61%

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Podestá¹,

Solano²,

Vedia

et al. 1984

European Journal of Biochemistry

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Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor -adrenal adenylate cyclase complexes were formed.Cell fusion has a great importance in the generation of heterocarions and hybrid cell lines [I -31. The technique has also been used to study the mobility of cell membrane markers and receptors [4, 51. Fusion of cells, in homogenous or heterogenous populations, is achieved in vitro by treating cell suspensions with inactivated sendai virus, poly(ethy1ene glycol) or lysophosphatidylcholine [2, 6-81. A main limitation of these 'fusogenic agents' is however, the low yield and the scarce viability of the hybrids thus obtained.Electrical-field-induced cell fusion has been recently proposed as an alternative to the use of the above-mentioned fusogenic agents [9 -111. The technique involves the orientation of cells under an asymmetric, alternating current field, a phenomenon called dielectrophoresis [I 2 -171 and the induction of the fusion process by an electrical breakdown pulse [9 -111. Evidence indicates that, with such a fusion procedure, yields of viable hybrids are several orders of magnitude higher than with methods using chemical fusogenic agents [9, 101. Since the electrofusion procedure has been only recently introduced to studies on animal cells [lo], there is not enough evidence available on the possibility of producing, with these methods, cell hybrids which can maintain highly specialized biochemical processes. For Friend cells it has been shown that highly specialized biochemical processes such as stimulation of haemoglobin synthesis in the presence of dimethyl sulfoxide, can be induced in electrofused cells [IS, 191. This paper shows that electrofusion of rat adrenal and Leydig cells produced hybrids bearing the capacity to be stimulated by lutropin or adrenocorticotropin, thus synthesizing both testosterone and corticosterone. MATERIALS AND METHODSLeydig cells were prepared by collagenase digestion of adult rat testis as described [20-211, and rat adrenocortical cells from the zona fasciculata-reticularis, were obtained by trypsin digestion [22]. The cells were resuspended in 0.3 M mannitol and electrofused by a modification of published procedures [9, 101. Fusion was performed on a perspex cylindrical chamber, having two parallel platinum electrodes (40 pm separating space). The chamber was mounted on a glass slide to allow microscope observation of pearl-chain formation and cell fusion. Two electrode wires were connected in parallel to a function generator (Health, model IG-1271), and to a home-made pulse generator. The first one was used to produce a high-frequency alternating field, resulting in cell dielectrophoresis and pearl-chain formation as described elsewhere [9, 10,12-141. The second one allowed the application of short constant voltage pulses to induce...

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Section: Resultssupporting

confidence: 61%

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Podestá¹,

Solano²,

Vedia

et al. 1984

European Journal of Biochemistry

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“…In the Leydig cells of the testis, there is a marked disparity between the large cyclic AMP response of intact cells to luteinizing hormone (LH) or human chorionic gonadotropin (hCG) (10,11) and the relatively poor response of adenylate cyclase in Leydig cell membranes (12). Also, there has been no published description of LH-induced activation of adenylate cyclase in rat Leydig cell membranes, although effects of LH on the ovarian enzyme have been readily demonstrable (13).…”

mentioning

confidence: 99%

Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell.

Dufau¹,

Báukal²,

Catt³

1980

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism by which peptide hormones stimulate adenylate cyclase is now recognized to involve an essential GTPdependent step (1). Since the original demonstration of the importance of guanyl nucleotides in hormone action, numerous tissues have been shown to be dependent on GTP for full expression of hormone-stimulated adenylate cyclase. The mechanism by which guanyl nucleotides influence adenylate cyclase enzyme is complex and includes interaction of the catalytic enzyme unit with a nucleotide-binding protein (2-8). The molecular interactions induced by agonist occupancy of 3-adrenergic receptors lead to the formation of a receptorguanyl nucleotide regulatory complex, indicating that both physical and functional coupling are required for catecholamine stimulation of adenylate cyclase activity (9). However, a direct demonstration of the hormone-stimulated nucleotide binding process has not been reported.

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“…Specific binding of bungarotoxin (8), peptide hormones (4,9), and f3-adrenergic receptor blocking agents (2,3) to solubilized membrane components containing the respective receptors has been demonstrated. Some success in reconstitution experiments has also been reported (8)(9)(10), but, even in these instances, evidence of actual solubilization (namely, complete disintegration of the original membrane structure) is often lacking. Expression of receptor function apparently requires the fragile interaction of several components and therefore damage by the solubilizing agent or its insufficient removal from only one of the components could prevent reconstruction of function.…”

mentioning

confidence: 98%

Functional implantation of a solubilized β-adrenergic receptor in the membrane of a cell

Eimerl

Neufeld

Korner

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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When the fi-adrenergic receptor of turkey erythrocytes was solubilized by deoxycholate, it retained its potential to activate an adenylate cyclase system. Electron microscopy showed that true solubilization had apparently been achieved; no residual membrane or vesicle structure was found. A different approach was therefore developed on the basis of earlier work (11, 12) on the hormone receptors that activate adenylate cyclase. It had been demonstrated that these receptors exist in the cell membrane as independent units. Furthermore, the receptors for different hormones from different cells were interchangeable; the receptor from one cell membrane could be coupled to the adenylate cyclase of another cell membrane by membrane fusion (11). On the basis of this information, for the present work the f3-adrenergic receptor was solubilized without regard for the other components of the adenylate cyclase system. After removal of the solubilizing agent the receptor was adsorbed to a phospholipid vehicle and implanted in an intact cell by a modification of a recently developed procedure (11). The receiving cell membrane had high adenylate cyclase activity and thus contained all the components of the adenylate cyclase system but no f3-adrenergic receptor (12). With the aid of this design, reestablishment of function of a solubilized fl-adrenergic receptor was readily obtained. * EXPERIMENTAL Cell Preparations. Friend erythroleukemia cells (Fc) have been described (12). Turkey erythrocyte membranes were prepared by the following modification of the procedure for frog erythrocytes (14). To the washed pellet of cells, DNase was added (1600 units/ml of packed cells) and the slurry was quickly poured into 20 vol of lysis medium (10 mM Tris, pH 8.0/2 mM mercaptoethanol/0.2 mM MgCl2) at 230C. After 20 min, the cell membranes were washed thrice at 4VC in 10 mM Tris, pH 8.0/0.1 mM ethylene glycol bis(f3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/2 mM mercaptoethanol.Solubilization of P-Adrenergic Receptor by Deoxycholate (Doc). Turkey erythrocyte membranes were washed and suspended in (3 mg/ml) 10 mM 3-(N-morpholino)propanesulfonate (Mops), pH 7.5/1 mM mercaptoethanol. The membranes were treated with N-ethylmaleimide (MalNEt) in the above medium to inactivate the adenylate cyclase (11). After they were washed, the membranes (2 mg/ml) were incubated in the same medium with 6 mM MgCl2 and 20,gM isoproterenol for 15 min at 300C. (11) for fusion of liver membranes with Fc cells, but Ca2+ was omitted and Mg2+ was 4 mM. Incubation was at 370C and dilution was terminated after the cumulative addition of 10.5 ml of medium. The term "implantation" will be used whenever the solubilized resedimented receptor interacted with a cell; the term "fusion" will be used for cell-cell and native membrane-cell interactions.Determination of Adenylate Cyclase and fl-AdrenergicReceptor Binding Sites. All operations were at 4VC. After receptor implantation, the cells (which were in the process of lysis) were centrifuged for' 10 min at 20,...

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Gonadal LH Receptors: Resolution from Adenylate Cyclase and Transfer to Heterologous Cells

Cited by 3 publications

References 6 publications

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell.

Functional implantation of a solubilized β-adrenergic receptor in the membrane of a cell

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