Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells.

Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of Gq (Ad-EF1-dnG alpha q) and the other expressing the wild-type alpha-subunit of Gq (Ad-EF1-wtG alpha q). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+]i) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha q inhibits the increase in [Ca2+]i produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha q, which indicates that in both HSG and HT29 cells, the increase in [Ca2+]i produced by muscarinic receptor activation is largely mediated by activation of Gq. Neither virus affected the resting level of [Ca2+]i in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.

Interaction of diazoxide and cromakalim with ATP-regulated K+ channels in rodent and clonal insulin-secreting cells.

The hyperglycaemia-inducing sulphonamide diazoxide has been previously shown to mediate its effects upon insulin secretion by opening K+ channels and hyperpolarizing the beta-cell membrane. The target site has been characterized as the ATP-regulated K+ (K+ATP) channel protein. In the present study, a detailed investigation of interactions of diazoxide and another K+ channel opener, cromakalim, with K+ATP channels has been performed in individual insulin-secreting cells using patch-clamp techniques. In agreement with previous studies, diazoxide and cromakalim were found to be effective only when ATP was present upon the inside face of the plasma membrane. The ability of both diazoxide and cromakalim to open channels was, however, found to diminish with time following isolation of inside-out patches. Within seconds of forming the recording configuration, the actions of both compounds were potent, and were found to decline steadily as the number of operational channels decreased ('run-down'). In open cells, where the plasma membrane remains partially intact, the rate of run-down was significantly reduced, and effects of channel openers were recorded up to 80 min following cell permeabilization. We also demonstrated that in the absence of ATP, but in the presence of ADP, both diazoxide and cromakalim were able to open K+ATP channels. Interestingly, once the effects of diazoxide and cromakalim on K+ATP channels in the presence of ATP were lost, both compounds opened channels in the presence of ADP. One implication of these data is that the actions of diazoxide and cromakalim involve regulatory proteins associated with the ion channel; this molecule is able to bind ATP, ADP and possibly other cytosolic nucleotides.

Polymyxin B has multiple blocking actions on the ATP-sensitive potassium channel in insulin-secreting cells.

The action of polymyxin B (0.1 microM) on ATP-sensitive K+ (K+ATP) channels in RINm5F insulin-secreting cells was investigated by patch-clamp techniques. Using inside-out patches, open-cells and outside-out patches, polymyxin B was found to block K+ATP channels by, on average, approximately 90-95% of the initial control level of channel activity. The effects were rapid in onset, sustained and readily reversible. Similar effects were found in patches excised from cells pretreated overnight with 1 microM of the phorbol ester phorbol myristate acetate (PMA). External block of channels was associated with a marked decrease in single-channel current amplitude, whereas these effects were not seen when polymyxin B was added to the inside face of the membrane. In patches bathed with internally applied ATP (0.5 mM) and ADP (0.5 mM), polymyxin B inhibited channels but its actions were not reversible upon removal of the compound. However, when the same protocol was undertaken upon cells pre-treated with PMA, the effects of polymyxin B were readily reversed. Our data suggests that polymyxin B is a novel modulator of K+ATP channels, exhibiting multiple blocking actions that may possibly involve a direct effect upon the channel and indirect effects mediated through the inhibition of endogenous protein kinase(s).

Properties of diazoxide and cromakalim-induced activation of potassium channels in cultured rat and RINm5F insulin-secreting cells; effects of GTP.

Experiments have been carried out to examine the effects of GTP on the opening of K+ channels in insulin-secreting cells by diazoxide (0.2 mM) and cromakalim (0.5 mM). Using rat pancreatic beta-cells and RINm5F insulinoma cells, patch-clamp recordings of unitary ATP-sensitive potassium (K+ATP) channel currents were made in either the open cell or outside-out patch recording configurations. Adding diazoxide or cromakalim to either the inside or the outside face of the membrane was found regularly to cause the activation of K+ATP channels in the presence of 0.5 mM ATP. We now demonstrate that in the absence of ATP but in the presence of GTP (0.5-1 mM), both diazoxide and cromakalim activate channels. Effects are rapid in onset, sustained and readily reversible. Both the diazoxide- and cromakalim-induced activation of K+ATP channels were mediated by increases in channel open-state probability, and were not associated with any significant change in either channel amplitude or by an increase in the number of channels in the patch. The actions of both diazoxide and cromakalim were not affected by overnight pretreatment of cells with pertussis toxin, suggesting that PTX-sensitive GTP-binding proteins are not involved in mediating the actions of either compound. These data indicate that diazoxide and cromakalim open K+ATP channels in a manner not solely dependent upon intracellular ATP, but by mechanisms involving other cytosolic nucleotides, including GTP.

Developmental changes in the management of acid loads during preimplantation mouse development.

Intracellular pH recovery in Quackenbush Swiss mouse preimplantation embryos following acid loading was investigated under conditions of H+-monocarboxylate cotransporter inactivity. Isoform-sensitive inhibitors of Na+-H+ exchange (NHE) were used to block the Na+-dependent component of the response. A biphasic dose-response curve for HOE-694 and N-methylisopropylamiloride (MIA) suggested that two isoforms (putatively NHE1 and NHE3) are active in the oocyte, 1-cell, and 2-cell stages. By the blastocyst stage, loss of one of the MIA-sensitive NHE activities (putatively NHE3) was observed in isolated inner cell masses, and an MIA-resistant component of the recovery was identified. The MIA-resistant component was inhibited by 2 mM amiloride and enhanced by external K+ and by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, suggesting NHE4 activity. However, unlike NHE4 in other tissues, the MIA-resistant component did not transport Li+ in exchange for H+, and reverse transcription-polymerase chain reaction detected NHE4 mRNA in the oocyte but not in later stages. Trophoblast, whether in intact or collapsed blastocysts, did not show measurable NHE activity or MIA-sensitive activity during recovery from acid load. Both trophoblast and pluriblast manifested an H+ conductance in response to acid load. This H+ conductance was first detected at the 8-cell stage and was blocked by zinc in the isolated inner cell mass but not in trophoblast. No other effective inhibitors of its activity were found.

The activity of the H+-monocarboxylate cotransporter during pre-implantation development in the mouse.

We have reported previously that cytosolic pH (pHi) in the mouse 2-cell conceptus is controlled by a H+-monocarboxylate cotransporter (MCT) that is sensitive to cinnamates and p-chloromercuriphenylsulfonate. In the present study we have used measurement of pHi with BCECF to characterize the changes in MCT activity during pre-implantation development. We found that the resting pHi in bicarbonate-free conditions increased significantly from the unfertilized oocyte to the 2-cell stage, but thereafter remained constant. There was no evidence for changes in MCT activity during the cell cycle, but MCT activity was found to increase during development. Using RT-PCR we demonstrated that mRNA encoding MCT isoforms 1, 2 and 3 is present throughout pre-implantation development. The inhibitor of MCT1, p-chloromercuribenzoic acid, completely abolished the effect of extracellular l-lactate on pHi suggesting that MCT1, and not MCT2, plays a functional role in pHi regulation in mouse conceptuses, while the role of MCT3 remains unclear. We further found that removal of glucose from the culture medium, which has previously been shown to stimulate pyruvate uptake by blastocysts, had no effect on the activity of the MCT. These findings suggest that the changes in pyruvate uptake that have been observed following compaction are not due to changes in the activity of the MCT. These findings indicate the presence of MCTs during early embryonic development.