31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

Dissociation of cellular K+ accumulation from net Na+ transport by toad urinary bladder

Summary A number of published data suggest a variable stoichiometry between the rates of cellular potassium uptake and net sodium transport (J _Na) across the urinary bladder of the toad. This problem was examined by simultaneously studying the intracellular chemical activity of potassium (a _K) with open-tip K⁺-selective microelectrodes and micropipets, and monitoringJ _Na by measuring the short-circuit current (SCC). When bathed in the short-circuited state with solutions containing ana _K of 2.7mm, the mean ±sem values for intracellulara _K were 43±0.6mm.Ouabain, at a concentration of 10^–2 m, reduced intracellulara _K by 56–67% and SCC by 96–100%. At 5×10^–4 m, ouabain reversibly reduced intracellulara _K by 40–55%, and SCC by 63–68%; the inhibition of SCC was only partly reversible during the period of observation.Removal of external potassium reduced intracellulara _K by 69–80% and SCC by 51–76%. Restoration of external potassium entirely returned intracellulara _K to its control value, but only partially reversed the inhibition of SCC during the period of study. Furthermore, recovery ofa _K began 19–43 min before that of SCC; recovery ofa _K was 90–97% complete before any increase in SCC could be measured. Although other interpretations are possible, the simplest interpretation of the data is that the processes responsible for potassium accumulation and transepithelial sodium transport are not identical. We propose the existence of a separate transfer mechanism at the basolateral cell membrane, responsible for accumulating intracellular potassium, and not directly coupled to active sodium transport.     

Potassium activities in epithelia

In general, intracellular K+ appears to be compartmentalized. This phenomenon does not seem to characterize cytoplasm per se, but probably reflects the processes of sequestration and ion exclusion characterizing certain as yet unidentified organelles. The cell nucleus does not appear to participate significantly in these processes. Measurement of intracellular potassium activity (alpha K)c in small epithelial cells is complicated by significant technical problems. Recent experimental maneuvers designed to circumvent these problems have led to substantially higher estimates of (alpha K)c under baseline conditions. The time courses of short circuit current (SCC) and (alpha K)c in toad urinary bladder have been correlated under two experimental conditions. After removing external K+ or after adding ouabain, both parameters are depressed. However, the time courses of SCC and (alpha K)c are very different following return to baseline conditions. The data suggest: 1) that the processes of cell K+ accumulation and transepithelial Na+ transport are not linked with a fixed stoichiometry, and 2) if a reduction in cytosolic K+ activity does inhibit transepithelial Na+ transport, its role is indirect.