The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.     

Internal equilibrium of the hammerhead ribozyme is altered by the length of certain covalent cross-links

A method was developed that permits covalent cross-links of different linker lengths to be introduced into RNA at defined positions. The previous observation that a cross-link between stems I and II of the hammerhead ribozyme was confirmed and further explored. By examining the catalytic consequences of varying the position and length of this cross-link, we conclude that the previously proposed conformational dampening model cannot sufficiently explain the increase in ligation rate induced by the cross-link. Rather, the cross-link constrains the cleaved hammerhead into a structure that more closely resembles the transition state, thereby increasing the reverse ligation rate relative to a non-cross-linked control.     

Ethnic variation in tyrosinase and TYRP1 expression in photoexposed and photoprotected human skin

The relative expression of a number of key mediators of human pigmentation including tyrosinase, tyrosinase related protein-1 (TYRP1), endothelin-1 and adrenocorticotrophic hormone (ACTH) proteins were analysed and quantified in immunohistochemically stained skin sections using semiquantitative computer assisted image analysis. Comparisons were made between a range of different ethnic skin types including European, Chinese, Mexican, Indian and African at both chronically photoexposed and photoprotected sites. Melanocyte number varied little with ethnicity except in the European group which had 60-80% more melanocytes than other skin types (P < 0.01, n = 10; Student Neuman-Keuls). However, melanocyte number was increased approximately twofold in chronically photoexposed skin of all ethnic groups (P < 0.001, n = 48; paired t-test). Tyrosinase protein expression in melanocytes did not vary with ethnicity, but TYRP1 protein was significantly elevated (approximately 2.6-fold) in darkly pigmented African and Indian skin types compared with lightly pigmented Mexican, Chinese and European skin types. In melanocytes from chronically photoexposed skin, there was a modest but significant increase in the expression of tyrosinase protein (approximately 1.2-fold, P < 0.001, n = 48; paired t-test), together with a significant and slightly larger increase in the expression of TYRP1 protein (approximately 1.6-fold, P < 0.005, n = 48; paired t-test). In contrast, the expression of endothelin-1 and ACTH showed no significant variation with either ethnicity or photoexposure. These data are consistent with the view that maintenance of a chronically hyperpigmented phenotype in chronically photoexposed human skin is largely the result of a stable increase in the number of tyrosinase positive melanocytes at these sites. Moreover, the observed ethnic variation in TYRP1 protein expression suggests that TYRP1 may play a significant role in mediating ethnic differences in melanogenesis and constitutive skin pigmentation in vivo.     

Correlating a protein structure with function of a bacterial mechanosensitive channel

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.     

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa. impacts on lignin structure and implications for the biosynthesis of G and S lignin

Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in beta-5 linked dimers of G units. Accumulation of soluble caffeic acid beta-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis.