### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

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The scanning model for translation: an update.

GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.

The GTPase superfamily: conserved structure and molecular mechanism

The protective effects of estrogen on the cardiovascular system

Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodeling, and the localization of atherosclerotic lesions. Major regulation of the blood vessel responses occurs by the action of hemodynamic shear stresses on the endothelium. The transmission of hemodynamic forces throughout the endothelium and the mechanotransduction mechanisms that lead to biophysical, biochemical, and gene regulatory responses of endothelial cells to hemodynamic shear stresses are reviewed.

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Flow-mediated endothelial mechanotransduction

Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries.

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultured endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with Kd of 7.2 +/- 3.5 nM and maximal binding (Bmax) of 121.4 +/- 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; Ki = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and the combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were ADP-ribosylated by pertussis toxin. The labelling intensity was Gi alpha 2>Gi alpha 3-Gi alpha l and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5-HT (3 x 10-6 M; 4 min) decreased significantly further ADP-ribosylation of Gi alpha 2 by pertussis toxin, but not that of Gi alpha 3 and/or Gi alpha l.5. The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Gi alpha l protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Gi alpha proteins or the absence of 5-HT binding sites.

Gi proteins and the response to 5‐hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF

Purification of G proteins

1. Four questions raised by previous studies that had shown activation of K+ channels by alpha subunits of the type 3 Gi protein are addressed in the present communication: a) are K+ channels specific for one Gi? b) are there more ionic channels under direct G protein control? c) can we confirm using recombinant G alpha s the results obtained with biochemically resolved G alpha s and continue ascribing the regulatory effector to this part of the alpha beta gamma holo-G protein? and d) can we confirm that a single G alpha, Gs alpha in this case, is able to affect more than one type of effector function? 2. We found Gi alpha s are isoforms, that there exist also Gi-insensitive, Go-responsive K+ channels and that G alpha s can be multifunctional. Thus, a single receptor will elicit cellular responses that will depend on the endogenous G protein as well as the type of effector function expressed in it. 3. In another set of experiments we found that G beta gamma s, be they derived from human erythrocytes, human placenta, bovine brain or bovine retina, all inhibit Gk-gated K+ channel activity as seen in inside out membrane patches with GTP as the driving nucleotide. In addition we noted that inhibition was much more effective under basal (no agonist in the pipette) than agonist stimulated conditions, as reported in earlier experiments in which beta-adrenoceptors, Gs and catalytic unit of adenylyl cyclase had been incorporated into phospholipid vesicles. 4. We propose that one of the roles of G beta gamma s in membranes is to quench ligand independent G protein activation by unoccupied receptors. Other roles of G beta gamma s are: a) by re-associating with GDP-G alpha s, to promote interaction with receptors, and b) by dissociating from activated R.G alpha *GTP.beta gamma, to allow for receptor dissociation from GTP-activated G alpha s, which is required to satisfy the catalytic mode of receptor action.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2125.1990.tb05463.x

G protein coupling of receptors to ionic channels and other effector systems.

The central role of G proteins in coupling receptors to effector systems can be best illustrated by the contents of Table 1, which lists receptors that exert their actions by interacting with a G protein, according to the general schemes shown in Fig. 1. Like receptors, which are increasing in number rapidly, effectors affected by the activated forms of G proteins are also increasing, most notably through the discovery in 1986–1987 that ionic channels form part of the family of molecules regulated by G proteins. Using cell-free systems such as provided by excision of membrane patches from cells and incorporation of plasma membrane vesicles into lipid bilayers, it was shown that ionic channels are indeed regulated by activated G proteins. Some of these channels had long before been predicted to be under the control of G-protein-coupled receptors by means other than soluble second messengers. The mechanism by which G proteins regulate some of these channels is at the very least “membrane delimited” and independent of any phosphorylation event or of changes in cytoplasmic levels of second messengers such as cAMP, Ca2+, or IP3, and is very likely due to direct interaction of the G protein α-subunit and the channel proper (for review, see Brown and Birnbaumer 1988). Our group was prominent in providing some of the initial as well as subsequent supporting data for these conclusions (Brown and Birnbaumer 1988; Yatani et al. 1987a,b,c, 1988a,b,c; Codina et al. 1987a,b, 1988; Imoto et al. 1988; Kirsch et al. 1988). Another research group has claimed that ionic channels, specifically the heart muscarinic K+ channel, now referred to as Gi-gated K+ channel (see below), may be regulated by receptors, not via a specific G protein α-subunit, but via the βγ-subunits of G proteins (Logothetis et al. 1987).

Juan Codina

Papers

Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries.

Gi proteins and the response to 5‐hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF

Purification of G proteins

G protein coupling of receptors to ionic channels and other effector systems.

Multiple Roles of G Proteins in Coupling of Receptors to Ionic Channels and Other Effectors