Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Reproductive strategies in the freshwater sphaeriid clam,Musculium partumeium (say), from a permanent and a temporary pond

Summary Reproduction was studied in two populations ofMusculium partumeium from temporary and permanent ponds. Adults of the single annual generation from the ephemeral pond have an annual selection ratio of 25:1 with 37.03 gC per newborn, and an intrinsic rate of increase (r) of 0.0084 day^-1. Fall-born adults from the permanent pond have an annual selection ratio of 38:1, 21.82 gC per newborn and anr of 0.0115 day^-1; springborn adults have an annual selection ratio of 136:1 (107:1 for their contribution to fall birth and 29:1 for the spring birth period) with newborns of 24.21 gC and anr of 0.0304. The trade off between quantity and quality of young is discussed in terms of adaptive strategies.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology

Summary This is the third in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxribonucleate)-3-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article describes the structure of the nuclease and of a nuclease-inhibitor complex as determined by x-ray crystallography. The crystal structures are correlated with some of the known chemical and enzymological properties of the enzyme, and the three areas combined to propose a mechanism of action.This article is the third in a series of four devoted to the stapholoccal nuclease. Reviews concerning its isolation and enzymology (1) as well as the features of its ligand binding site (2) have appeared in previous issues. Work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, NIH and from the Robert A. Welch Foundation to F. A. Cotton and E. E. Hazen, Jr.     

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart.

Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

The effect of osmotic stress on the oxidation of glycolate by the blue-green alga Anacystis nidulans

Planta - Anacystis nidulans Richt. was shown to assimilate glycolic acid, and uptake was light-stimulated. In the dark 90% of the glycolate taken up was oxidised to CO2. Both light and dark uptake...     

A phenomenological computational model of the evoked action potential fitted to human cochlear implant responses

There is a growing interest in biomedical engineering in developing procedures that provide accurate simulations of the neural response to electrical stimulus produced by implants. Moreover, recent research focuses on models that take into account individual patient characteristics.We present a phenomenological computational model that is customized with the patient’s data provided by the electrically evoked compound action potential (ECAP) for simulating the neural response to electrical stimulus produced by the electrodes of cochlear implants (CIs). The model links the input currents of the electrodes to the simulated ECAP.Potentials and currents are calculated by solving the quasi-static approximation of the Maxwell equations with the finite element method (FEM). In ECAPs recording, an active electrode generates a current that elicits action potentials in the surrounding auditory nerve fibers (ANFs). The sum of these action potentials is registered by other nearby electrode. Our computational model emulates this phenomenon introducing a set of line current sources replacing the ANFs by a set of virtual neurons (VNs). To fit the ECAP amplitudes we assign a suitable weight to each VN related with the probability of an ANF to be excited. This probability is expressed by a cumulative beta distribution parameterized by two shape parameters that are calculated by means of a differential evolution algorithm (DE). Being the weights function of the current density, any change in the design of the CI affecting the current density produces changes in the weights and, therefore, in the simulated ECAP, which confers to our model a predictive capacity.The results of the validation with ECAP data from two patients are presented, achieving a satisfactory fit of the experimental data with those provided by the proposed computational model.