Brain region-specific and time-dependent decreases of G alpha i2 in amygdala-kindled rats.

Immunoreactivity levels of G protein alpha-subunits, Galphas (45 and 52 kDa), Galphai1, and Galphai2, were determined in plasma membranes prepared from ipsilateral amygdala-pyriform cortex (AM/PC), contralateral AM/PC, dorsal hippocampus (DH), and ventral hippocampus (VH) in amygdala-kindled rats 24 h, 1 month, and 3 months after the last seizure. Relative to sham controls, immunoreactivity levels of Galphai2 were significantly reduced in ipsilateral AM/PC and DH, with maximal decreases observed, respectively, at 24 h (26% form control levels) and 1 month (18% from control levels) postseizure. No significant alterations in the levels of Galphai1 or either of the Galphas isoforms were observed. The reduction in Galphai2 levels was, however, not accompanied by measurable changes in Galphai-mediated inhibition of adenylyl cyclase, as estimated by GppNHp modulation of forskolin-activated adenylyl cyclase activity. The present findings add support to the hypothesis that long-term changes in brain functioning following kindling may involve altered expression and/or function of components of the transmembrane signalling cascade, including G proteins.

Substance P in intervertebral discs. Binding sites on vascular endothelium of the human annulus fibrosus.

The annulus fibrosus of the human intervertebral disc is sparsely innervated, some of the fibers containing substance P. We could demonstrate, by autoradiography, binding sites for substance P localized on the endothelium of small blood vessels in the annulus fibrosus of human intervertebral discs removed during anterior fusion for back pain. In binding inhibition studies, binding of 125I-Bolton Hunter-substance P was inhibited by unlabeled substance P and the related tachykinins neurokinin A and neurokinin B with a rank order of potency substance P > NKA > NKB. Specific binding was reduced > 75 percent by 5'-guanylylimidodiphosphate, indicating G-protein coupling. These features are characteristic of an NK1 receptor through which vascular effects, i.e., vasodilation, plasma extravasation and angiogenesis of substance P, are mediated. The presence of NK1 receptors on blood vessels in the annulus fibrosus may indicate a role for substance P in tissue repair although acute proinflammatory effects may contribute to discogenic pain.

Effect of guanine nucleotides and temperature on calcitonin gene-related peptide receptor binding sites in brain and peripheral tissues.

Recent data have suggested the existence of at least two major classes of calcitonin gene-related peptide (CGRP) receptors in brain and peripheral tissues [Henke et al., Brain Res., 410 (1987) 404-408; Dennis et al., J. Pharmacol. Exp. Ther., 251 (1989) 718-725; ibid, 254 (1990) 123-128; Quirion et al., Ann. NY Acad. Sci., 657 (1992) 88-105]. However, little is currently known in the structure characteristics of CGRP receptors as cloning as yet to be reported. In the present study, the sensitivity of [125I]humanCGRP alpha binding to guanine nucleotides and temperature was investigated in guinea pig atria (prototypical CGRP1 tissue) guinea pig vas deferens (prototypical CGRP2 tissue) and in the rat brain and cerebellum (mixed assay). Binding isotherms of [125I]hCGRP alpha in those four tissue preparations were curvilinear and best fitted to a two-site model under most assay conditions. The high affinity binding component was highly temperature-sensitive and accounted, under experimental conditions, for up to 18% of the total population of receptors. Moreover, these high affinity sites were also highly sensitive to guanine nucleotides (Gpp(NH)p, 100 microM) in all preparations although to a different extend depending upon assay temperatures. Taken together, this suggests that the different CGRP receptor subtypes present in these tissue all belong to a G-protein coupled receptor family.

Role of histidine residues in agonist and antagonist binding sites of A1 adenosine receptor.

The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N6-[adenine-2,8-3H, ethyl-2-3H]phenylisopropyladenosine ([3H]R-PIA) and antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-independent effects of muscarinic antagonists on Ca2+ and K+ currents in frog and rat cardiac cells.

1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of beta-adrenergic receptors in dispersed rat testicular interstitial cells.

Recent studies have shown that beta-adrenergic agents stimulate steroidogenesis and cyclic AMP formation in mouse Leydig cells in culture. To obtain information about the possible presence and the characteristics of a beta-adrenergic receptor in rat testicular interstitial cells, the potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) was used as ligand. Interstitial cells prepared by collagenase dispersion from rat testis were incubated with the ligand for 2 h at room temperature. [125I]cyanopindolol binds to a single class of high affinity sites at an apparent KD value of 15 pM. A number of sites of 6,600 sites/cell is measured when 0.1 microM (-) propranolol is used to determine non-specific binding. The order of potency of a series of agonists competing for [125I]cyanopindolol binding is consistent with the interaction of a beta 2-subtype receptor: zinterol greater than (-) isoproterenol greater than (-) epinephrine = salbutamol much greater than (-) norepinephrine. In addition, it was observed that the potency of a large series of specific beta 1 and beta 2 synthetic compounds for displacing [125I]cyanopindolol in rat interstitial cells is similar to the potency observed for these compounds in a typical beta 2-adrenergic tissue, the rat lung. For example, the potency of zinterol, a specific beta 2-adrenergic agonist, is 10 times higher in interstitial cells and lung than in rat heart, a typical beta 1-adrenergic tissue. Inversely, practolol, a typical beta 1-antagonist, is about 50 times more potent in rat heart than in interstitial cells and lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Initiation factors eIF4A and C1 from wheat germ and the formation of mRNA X ribosome complexes.

The binding of ribosomes to mRNA is analyzed in a fractionated system from wheat germ with [3H]uridine-labeled poly(A)+ RNA prepared from germinating wheat embryos. The reaction requires factors eIF3, eIF4C, and eIF5; Met-tRNA and the Met-tRNA binding system; either GTP or GMP-PNP; ATP; and factors C1 and eIF4A. These requirements are identical to those previously found to be necessary for formation of ribosome X Met-tRNAMeti complexes, with the exception of ATP, and factors C1 and eIF4A. The function of factors C1 and eIF4A is therefore specifically related to the mRNA attachment reaction. The presence of GTP in the mRNA binding reaction results in the formation of 80 S ribosome complexes, while with GMP-PNP only 40 S ribosome complexes are formed. Ribosome binding to native reovirus RNA in the fractionated wheat germ system is similar to the reaction with poly(A)+ RNA, strongly requiring ATP and factors C1 and eIF4A. Binding to inosine-substituted reovirus RNA, however, is only partially dependent upon ATP, and both the ATP-dependent and the ATP-independent binding reactions strongly require factor C1 and are substantially stimulated by factor eIF4A. The ATP-independent reaction is inhibited by pm7GDP, has a strong requirement for Met-tRNAMeti, and the 40 S ribosome complex is stable to RNase. These results indicate that the ATP-independent binding of ribosomes to inosine-substituted reovirus RNA proceeds through the normal initiation process. They further suggest that neither factor C1 nor eIF4A function exclusively to unwind mRNA secondary structure. Since eIF4A is required for the ATP-independent binding to inosine mRNA, and at the same time interacts with ATP in the reaction with ATP-requiring mRNAs, this factor may have two roles in protein chain initiation, one related to the mRNA X ribosome interaction, and one related to the function of ATP.

Effects of nucleoside triphosphates on choleragen-activated brain adenylate cyclase.

To investigate the effects of nucleoside triphosphates on the activation of adenylate cyclase by choleragen and on the stability and catalytic function of the choleragen-activated enzyme, we treated samples of particulate preparation from bovine brain successively in three separate incubations with extensive washing between each step. In incubation I, choleragen and NAD were pesent to activte the adenylate cyclase. In incubation II, conditions were varied to assess enzyme stability. Finally, adenylate cyclase activity was assayed with ATP or adenylyl imidodiphosphate [App-(NH)p] as the substrate. Even when assays contained an optimal concentration of GTP, nucleoside triphosphate (plus a regenerating system) was required in incubation I for maximal choleragen activation; in order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. During incubation II (at 30 degrees C), activity of the choleragen-treated fractions was essentially completely stable when 100 microM GTP (plus a regenerating system) was present. ITP and ATP were less effective. Activation produced by guanylyl imidodiphosphate was more stable than that resulting from choleragen, GTP, and NAD. After activation of membranes with choleragen, NAD, and GTP, nucleoside triphosphate plus a regenerating system (but not NAD or additional choleragen) was essential for expression of maximal activity. In order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. It appears that GTP, which was effective in micromolar concentrations, plays an important role not only in the activation of adenylate cyclase by choleragen but also in the stabilization and expression of the catalytic function of the activated enzyme.