Participation of RGS8 in the ternary complex of agonist, receptor and G-protein.

The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein alpha subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K(+) channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and G(o) alpha(A)-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gbetagamma overexpression. FRET was also seen between RGS8 and Ggamma, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein alpha subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the 'kinetic efficacy' of G-protein activation.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-DeltaF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the betagal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca(2+)-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Human ClC-3 is not the swelling-activated chloride channel involved in cell volume regulation.

Volume regulation is essential for normal cell function. A key component of the cells' response to volume changes is the activation of a channel, which elicits characteristic chloride currents (I(Cl, Swell)). The molecular identity of this channel has been controversial. Most recently, ClC-3, a protein highly homologous to the ClC-4 and ClC-5 channel proteins, has been proposed as being responsible for I(Cl, Swell). Subsequently, however, other reports have suggested that ClC-3 may generate chloride currents with characteristics clearly distinct from I(Cl, Swell). Significantly different tissue distributions for ClC-3 have also been reported, and it has been suggested that two isoforms of ClC-3 may be expressed with differing functions. In this study we generated a series of cell lines expressing variants of ClC-3 to rigorously address the question of whether or not ClC-3 is responsible for I(Cl, Swell). The data demonstrate that ClC-3 is not responsible for I(Cl, Swell) and has no role in regulatory volume decrease, furthermore, ClC-3 is not activated by intracellular calcium and fails to elicit chloride currents under any conditions tested. Expression of ClC-3 was shown to be relatively tissue-specific, with high levels in the central nervous system and kidney, and in contrast to previous reports, is essentially absent from heart. This distribution is also inconsistent with the previous proposed role in cell volume regulation.

Murine CFTR channel and its role in regulatory volume decrease of small intestine crypts.

Cystic fibrosis (CF) is caused by mutations in the secretory Cl(-) channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl(-) channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other 'environmental' factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl(-) channel and as a regulator of volume-dependent homeostatic cell mechanisms.

History counts: a comparative analysis of racial/color categorization in US and Brazilian censuses.

Categories of race (ethnicity, color, or both) have appeared and continue to appear in the demographic censuses of numerous countries, including the United States and Brazil. Until recently, such categorization had largely escaped critical scrutiny, being viewed and treated as a technical procedure requiring little conceptual clarity or historical explanation. Recent political developments and methodological changes, in US censuses especially, have engendered a critical reexamination of both the comparative and the historical dimensions of categorization. The author presents a comparative analysis of the histories of racial/color categorization in American and Brazilian censuses and shows that racial (and color) categories have appeared in these censuses because of shifting ideas about race and the enduring power of these ideas as organizers of political, economic, and social life in both countries. These categories have not appeared simply as demographic markers. The author demonstrates that censuses are instruments at a state's disposal and are not simply detached registers of population and performance.

Modulation of the effects of extracellular ATP on [Ca2+]i in rat brain microvacular endothelial cells.

This study examined the intracellular regulation of signal transduction initiated by activation of the P2Y2 purinoceptor in a cultured rat brain microvascular endothelial cell line (RBE4). Intracellular free Ca2+ ([Ca2+]i) was monitored in single cells, using FURA-2 fluorimetry. As previously described [Nobles, M., Revest, P.A., Couraud, P.-O., Abbott, N.J., 1995. Characteristics of nucleotide receptors that cause elevation of cytoplasmic calcium in immortalized rat brain endothelial cells, RBE4, and in primary cultures. Br. J. Pharmacol., 115, 1245-1252], extracellular ATP (100 microM, 20 s) evoked a transient increase in intracellular free calcium concentration ([Ca2+]i). The amplitude of the Ca2+ transient evoked by ATP decreased with successive applications (desensitisation), as expected for a P2 purinoceptor. The modulation of the Ca2+ signal downstream to the activation of the ATP receptor was investigated, using agents selected for their ability to interfere with the intracellular pathways activated by ATP. The amplitude of the Ca2+ transient observed on the second application of ATP was compared in the presence and absence of these agents. The Ca2+ transient triggered by ATP was decreased by the inhibitor of nitric oxide synthesis, N-omega-nitro-L-arginine methyl ester (L-NOARG). The inhibition induced by 100 microM L-NOARG was reversed by coapplication of the permeant cGMP analogue 8-brcGMP (100 microM). 8-BrcGMP caused a transient increase in [Ca2+]i when applied alone, and a dose-dependent inhibition of the increase in [Ca2+]i elicited by ATP. Indomethacin, an inhibitor of prostaglandin synthesis, inhibited the response to ATP. The inhibition caused by 10 microM indomethacin was reversed by coapplication of the permeant analogue of cAMP, 8-brcAMP (100 microM). 8-BrcAMP caused a transient rise in [Ca2+]i when applied alone, and a dose-dependent inhibition of the Ca2+ response evoked by ATP. The non-permeant cyclic nucleotides cAMP and cGMP did not affect the desensitising response to ATP, nor did they reverse the inhibitory actions of L-NOARG or indomethacin. It is concluded that cyclic nucleotides, nitric oxide, and prostaglandin synthesis pathways are able to interact with the Ca2+ second messenger pathway in rat brain endothelial cells activated by extracellular ATP.

Decline of the calcium response on successive stimulation of a rat brain endothelial cell P2U purinoceptor.

A microfluorimetric method using Fura-2 as calcium indicator was used to study the mechanism of desensitization of the calcium response evoked by activation of a brain endothelial cell P2U receptor. The study was mainly carried out on an immortalized rat brain endothelial cell line (RBE4), with some additional experiments on primary cultured rat brain microvascular endothelial cells. As previously described (Nobles et al. 1995), ATP (100 microM, 20 s) caused a transient increase in intracellular calcium levels ([Ca2+]i). This effect was dependent on the rate of filling of intracellular calcium stores, since a large inhibition of the ATP-mediated response was seen in the presence of cyclopiazonic acid, an inhibitor of the store Ca(2+)-ATPase. Application of repeated pulses of extracellular ATP led to a desensitization of the response, as measured by a decline in the release of intracellular calcium (Nobles et al. 1995). This desensitization was partially reversed after 300 s of incubation in agonist-free medium. Extracellular phosphorylation of the purinergic receptor appeared not to be involved in the desensitization process, since a similar rate of desensitization was obtained with the non-hydrolysable ATP analogue ATP gammaS. Oxidation of the purinergic receptor cannot account for the desensitization, since the decline of the ATP-mediated response was unchanged in the presence of 3 mM dithiothreitol. In the presence of ATP together with UTP, two equally potent activators of the P2U receptor, the desensitization was less than in the presence of only one of the agonists. The desensitization was greater when ATP was applied for longer (150 s) periods. Although these results do not exclude the participation of post-receptor events in the desensitization process, they suggest that desensitization is governed at least in part by agonist-receptor interaction.

Characteristics of nucleotide receptors that cause elevation of cytoplasmic calcium in immortalized rat brain endothelial cells (RBE4) and in primary cultures.

1. A dual-wavelength microfluorimetric method using Fura-2 as calcium indicator was applied to cells from an immortalized cell line of rat brain microvascular endothelial cells (RBE4), and to primary cultured rat brain endothelial cells. 2. In RBE4 cells, a brief (20 s) pulse of extracellular ATP (100 microM) induced a transient increase in the cytoplasmic calcium level ([Ca2+]i). Control responses to 100 microM ATP consisted of a ratio increase of 0.64 +/- 0.03 (mean +/- s.e., n = 51). Responses were seen at a concentration of 2.5 microM and were maximal at 100-1000 microM. When extracellular calcium was chelated with EGTA, the transient increase in [Ca2+]i was not affected. The results are consistent with Ca2+ mobilization from intracellular stores. 3. The purinoceptor involved belongs to the P2 subtype, since the agonist potency order among the adenine nucleotides was ATP > ADP > AMP. Moreover, the increase in [Ca2+]i evoked by ATP was partially inhibited by the P2 antagonist, suramin but was not affected by 8-phenyltheophylline, a P1-purinoceptor antagonist. The strong desensitization observed with repeated applications of ATP is also typical of a P2 receptor. 4. 2-Methylthio-ATP (2meS-ATP 100 microM), a P2Y agonist, elevated [Ca2+]i in only 17% of the cells tested; however, 2meS-ATP was found to antagonize the effect of ATP in all cells tested. The increase in [Ca2+]i evoked by ATP was inhibited by 500 s application of the P2Y purinoceptor antagonist, Reactive Blue 2 at 10 microM, while 60 s application of 100 microM was ineffective. 5. The uracil nucleotide, UTP (100 microM) was as effective as ATP in increasing [Ca2+]i. The effects of ATP and UTP were not additive. Cells desensitized to the action of ATP (or UTP) were unable to respond to UTP (or ATP).6. alpha,beta Methylene-ATP (alpha,beta meATP 100 microM), a P2x, agonist, elevated [Ca2+], in only 40% of the cells tested. In these cells it was less effective than ATP in increasing [Ca2+]i.7. Cells desensitized to the action of ADP responded, to a smaller extent, to ATP. In contrast, cells desensitized to the action of ATP were unable to respond to ADP.8. On primary cultures of brain endothelial cells the increase in [Ca2+]i in response to extracellular ATP(100 microM) and UTP (100 microM) was of an equivalent amplitude, and similar to the response in RBE4 cells.The pattern of desensitization was also similar to that in RBE4 cells.9 This comparative study indicates that in well-characterized brain microvascular endothelial cells that retain brain endothelial characteristics, the major class of nucleotide receptor is of the P2mu type. The implications for physiology are discussed.

Activation of basolateral Cl- channels in the rat colonic epithelium during regulatory volume decrease.

Exposure to a hypotonic medium caused an increase in the diameter of isolated crypts from the rat colon. The increase in cell volume was only transient and lasted about 7 min. Despite of the continuous presence of the hypotonic medium, cell volume decreased again. This regulatory volume decrease (RVD) was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker, and by Ba2+, a K+ channel blocker. Cell-attached patch-clamp recordings revealed that the RVD was associated with the activation of previously silent basolateral channels. These channels were identified after excision of the patch as Cl- channels (28 pS) and as K+ channels (45-60 pS). The RVD was dependent on the presence of external Ca2+. The phospholipase A2 inhibitor, quinacrine, and the lipoxygenase blocker, nordihydroguaiaretic acid, inhibited RVD, while indomethacin had no effect. In Ussing chamber experiments an exposure to hypotonic media caused an initial, transient increase in tissue conductance (Gt), followed by a prolonged decrease in short-circuit current (Isc) and the potential difference (V). The height of the electrical response was dependent on the decrease in the osmolarity in a range from 20 mosmol l-1 to 90 mosmol l-1. The increase in Gt was blocked by NPPB and Ba2+, whereas the decrease in Isc or V was inhibited by NPPB but enhanced by Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the antisecretory action of prostaglandin D2 in the rat colon.

Previous studies have shown that prostaglandin D2 (PGD2) inhibits neuronally mediated secretion in the rat colon. This antisecretory action of PGD2 was further characterized by the use of a prostaglandin D receptor blocker. Prostaglandin D2 inhibited the neuronally mediated short-circuit current evoked by prostaglandin I2, which represents Cl- secretion. The concentration-response curve for the inhibition by PGD2 was shifted to the right in the presence of the prostaglandin D receptor blocker, AH 6809. AH 6809 had no effect on the short-circuit current response induced by prostaglandin E2 or iloprost, a stable prostaglandin I2 analogue, suggesting an interaction of the blocker with receptors specific for PGD2. A direct interaction of PGD2 with enteric neurones was studied by determining its effect on acetylcholine release from enteric neurones preloaded with [3H]choline. Prostaglandin D2 suppressed 3H release induced by electric field stimulation. It had, however, no effect on the release induced by depolarization with potassium. The results suggest that the inhibitory action of PGD2 on enteric cholinergic neurones is mediated by prostaglandin D receptors.

Segment-specific effects of the heat-stable enterotoxin of E. coli on electrolyte transport in the rat colon.

The heat-stable enterotoxin of E. coli (STa) induced an increase in short-circuit current (Isc) in the rat colon. The maximal increase in Isc was about three times larger in the proximal than the distal colon. The action of STa was mimicked by 8-Br-cyclic GMP. Unidirectional flux measurements revealed that STa decreased Na+ and Cl- absorption in the distal colon, while it decreased Na+ absorption and activated Cl- secretion in the proximal colon. In the distal, but not in the proximal colon, indomethacin inhibited the action of STa and of 8-Br-cyclic GMP. Inhibition by indomethacin could be overcome by addition of prostaglandin E2 or forskolin, but not by addition of a non-hydrolysable analogue of cyclic AMP, suggesting an action of STa on cyclic AMP hydrolysis. Amrinone and trequinsin, two inhibitors of cyclic GMP-inhibited phosphodiesterases, mimicked the action of STa on Isc and inhibited the response to a subsequent administration of the toxin indicating the modulation of a cyclic GMP-inhibited phosphodiesterase by STa in the distal colon. The results give evidence for different intracellular action sites of STa in the two parts of the rat colon.

Segmental heterogeneity of the rat colon in the response to activators of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway.

The electrolyte transport was compared in proximal and distal segments of the rat colon under control conditions and after induction of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway. Baseline short-circuit current was decreased by indomethacin and tetrodotoxin in the distal colon, indicating a spontaneous production of neuronally acting prostaglandins. In contrast, baseline short-circuit current in the proximal colon was decreased only by indomethacin, but not by tetrodotoxin. Unidirectional flux measurements revealed that in the distal colon sodium and chloride were absorbed, while the proximal colon secreted chloride. A morphological comparison between the distal and proximal epithelium revealed that the zonulae occludentes and the microvilli were longer in the distal colon. The size of the Golgi apparatus was several times larger in the crypt than in the surface region without differences between proximal and distal colon. Distal segments were more sensitive to an activator of the Ca(2+)-pathway, carbachol, or activators of the cAMP-pathway such as forskolin and a cAMP-analogue. In contrast, the activation of the cGMP-pathway by a cGMP-analogue or by the heat-stable enterotoxin of E. coli (STa) was more effective in the proximal colon. The results give evidence for a segmental specificity with regard to the intracellular pathways responsible for the activation of secretion.