Hormonal activation of single K+ channels via internal messenger in isolated pancreatic acinar cells

The mechanism underlying hormonal activation of potassium channels was investigated in pig pancreatic acinar cells by patch-clamp single-channel and whole-cell current recordings. It was shown directly that a peptide hormone belonging to the cholecystokinin-gastrin family, CCK5, can activate single voltage-sensitive potassium channels which can be blocked by tetraethylammonium. The single-channel currents were recorded from electrically isolated cell-attached membrane patches to which the hormone had no access and the activation must therefore involve an intracellular messenger. The hormonal response requires external Ca2+ in the isolated membrane-patch area indicating that calcium gating is not directly linked to hormone-receptor interaction.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

Monocyte suppression of antigen-specific lymphocyte responses in diffuse cutaneous leishmaniasis patients from the Dominican Republic

Patients from the Dominican Republic with diffuse cutaneous leishmaniasis showed in vivo and in vitro anergy to leishmanial antigen. Relatives of these DCL patients living in the same endemic area frequently showed skin test and lymphocyte reactivity to leishmanial antigens. This further supports the concept of specific anergy in patients with diffuse cutaneous leishmaniasis. Adherent suppressor cells modulate the antigen-specific lymphocyte proliferative response. Suppressor cells could also be isolated by Percoll gradient centrifugation. Co-culturing of lymphocytes and monocytes from HLA-identical leishmanin responders and nonresponders also identified the suppressor cell as a monocyte. In one patient, this suppression disappeared when clinical cure had been accomplished.     

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.     

The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.