Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization.     

The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion.

We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.     

Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast.

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels.     

The effects of salicylic acid on metabolism and potassium ion content in yeast.

Under anaerobic conditions, at low pH and 30 degrees, commercial baker's yeast loses K+ ion in the presence of salicylic acid. Glucose utilization is inhibited. In suspensions containing no glucose, carbohydrate stores of the cell are dissimilated to carbon dioxide and alcohol. The ion loss and inhibitory effects of salicylic acid on glucose utilization are reversed by washing the cells free of salicylate. The loss of K+ appears to be due at least partly to a K+-H+ exchange process. An unexplained maximum is seen in the curves of either net K+ loss or K+ efflux versus salicylic acid concentration. At 6 degrees the effects of salicylic acid on both endogenous metabolism and net K+ loss are minimal. Furthermore, no maximum is seen in the K+ loss-salicyclic concentration curve at this temperature. It is generalized that salicylic acid or salicylate may elicit K+ leakage from many types of cells, i.e., a fundamental action of this compound may be its ability to affect (reduce) K+ content of the cell; furthermore, it appears that the salicylate effects on K+ loss may be associated in an as-yet-unknown manner with the metabolic effects of this compound. The effects of salicylate on K+ loss in yeast may not be unique for this compound, since no experiments of this nature have been done with other penetrating undissociated acids.     

An energy-dependent efflux system for potassium ions in yeast

An efflux of potassium ions was demonstrated in mutants of yeast cells lacking a functional high affinity carrier system for monovalent cations. This efflux showed the following characteristics: (a) It was stimulated by the presence of a substrate, either glucose or ethanol. (b) It was stimulated by several cationic organic molecules, such as ethidium bromide, dihydrostreptomycin, diethylaminoethyldextran, and also by trivalent cations, such as Al3+ and lanthanides; this stimulation also depended on the presence of a substrate. (c) K+ efflux was decreased in yeast mutants with decreased ATPase activity, which generated a lower membrane potential. (d) Although the efflux appeared to be of an electrogenic nature, producing hyperpolarization of cells, it was accompanied by the efflux of phosphate, probably as an anion partially compensating for the large amount of cations leaving the cell. (e) K+ efflux was also accompanied by an uptake of protons. (f) The efflux appeared more clearly in cells grown in YPD medium, and not in more complex media nor in the same YPD medium if supplemented with Ca2+ or Mg2+. Efflux of monovalent cations produced by Tb3+ and organic cationic agents was also demonstrated in wild type strains. This efflux system appears to be, at least partially, electrogenic, but seems to be also an exchange system for protons and to function as a symport with phosphate; it may be involved in the regulation of the internal pH of the cell, and appears to be regulated by its link to the energetic status of the cell, probably through the membrane potential.     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.     

Yeast as a model organism to study transport and homeostasis of alkali metal cations

To maintain an optimum cytoplasmic K(+)/Na+ ratio, cells employ three distinct strategies: 1) strict discrimination among alkali metal cations at the level of influx, 2) efficient efflux of toxic cations from cells, and 3) selective sequestration of cations in organelles. Cation efflux and influx are mediated in cells by systems with different substrate specificities and diverse mechanisms, e.g. ATPases, symporters, antiporters, and channels. Simple eukaryotic yeast Saccharomyces cerevisiae cells proved to be an excellent model for studying the transport properties and physiological function of alkali-metal-cation transporters, and the existence of mutant strains lacking their own transport systems provided an efficient tool for a molecular study of alkali-metal-cation transporters from higher eukaryotes upon their expression in yeast cells.     

A putative K(+)-selective channel in the plasma membrane of yeast that is blocked by micromolar concentrations of external divalent cations and is insensitive to tetraethylammonium

At pH 7, addition of glucose to an anaerobic suspension of non-metabolizing yeast cells causes a transient net efflux of K+ from the cells and a concomitant transient hyperpolarization of the plasma membrane (Van de Mortel, J.B.J., et al. (1988) Biochem. Biophys. Acta 936, 421-428). Both phenomena are effectively suppressed in the presence of low concentrations of polyvalent cations. The concentrations of Mn2+, Ca2+, Ba2+, Mg2+, Sr2+ and La3+ required for half-maximal suppression of the transient hyperpolarization are 10, 17, 20, 38, 47 and 5 microM, respectively. Subsequent addition of EDTA 90 s after that of Ca2+ immediately restores both K+ efflux and cellular uptake of the fluorescent membrane potential probe 2-(dimethylaminostyryl)-1-ethylpyridinium (DMP). This suggests that an interaction of polyvalent cations with an external binding site blocks the putative K(+)-selective channel. Opening of this channel is not blocked by 20 mM tetraethylammonium nor by 100 microM 3,4-diaminopyridine. It is argued that this glucose-induced K(+)-conductive pathway is not identical to the voltage-gated K+ channels identified until now in patch-clamp studies of the yeast plasma membrane.     

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.     

Furosemide-sensitive potassium efflux in cultured mouse fibroblasts

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.     

Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase

Potassium transport coupled to ATP hydrolysis has been reconstituted in proteoliposomes using a highly purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe. The ATPase activity in the incorporated enzyme was strongly stimulated (2.2-fold) by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP). The H+/K+ exchanger nigericin (in the presence of K+) stimulated 1.6-fold the ATPase activity. When both ionophores were added together, the stimulation was increased up to 2.7-fold. When a potassium concentration gradient (high K+ in) was applied to the proteoliposome membrane, a significant drop in the CCCP-stimulated ATPase activity was observed. Inversion of the K+ concentration gradient (high K+ out) did not decrease the stimulation by CCCP. High Na+ in also decreased the stimulation induced by CCCP in the absence but not in the presence of external K+. However, high Li+ in had no effect. Direct potassium efflux from the proteolyposomes was detected upon addition of MgATP using a selective K+ electrode. The ATP-dependent potassium efflux was abolished in CCCP and/or nigericin-pretreated proteoliposomes. However, during steady state ATP hydrolysis, a transient and small K+ efflux was observed upon addition of a CCCP pulse. I propose that the plasma membrane Mg2+-dependent ATPase in yeast cells not only carries out electrogenic H+ ejection but also drives the uptake of potassium via a voltage-sensitive gate which is closed in the absence and open in the presence of the membrane potential.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons.

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 microM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/K+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ability of Sit4p to promote K+ efflux via Nha1p is modulated by Sap155p and Sap185p

We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomyces cerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of SAP185 increases efflux, whereas deletion decreases efflux. Two other homologs, SAP4 and SAP190, are without effect on K+ homeostasis. Both SAP155 and SAP185 require the presence of SIT4 for function, which encodes a PP2A-like phosphatase important for the G1-S transition through the cell cycle. Overexpression of either the outwardly rectifying K+ channel, Tok1p, or the putative plasma membrane K+/H+ antiporter, Kha1p, increases efflux in both wild-type and sit4Delta strains. However, overexpression of the Na+-K+/H+ antiporter, Nha1p, is without effect in a sit4Delta strain, suggesting that Sit4p signals to Nha1p. In summary, the combined activities of Sap155p and Sap185p appear to control the function of Nha1p in K+ homeostasis via Sit4p.     

Volume-dependent and NEM-stimulated K+,Cl- transport is elevated in oxygenated SS, SC and CC human red cells

Mechanisms involved in cell volume regulation are important in SS, SC cells as they might be involved in determining the extent of sickling and the generation of dense cells and irreversibly sickled cells. We have studied in these cells the response to cell swelling of the K+,Cl- transporter. We found that Hb SS, SC and CC red cells have higher values of a ouabain-resistant, chloride-dependent and NEM-stimulated K+ efflux than AA red cells. In contrast, the Na+,K+,Cl- cotransport estimated from the bumetanide-sensitive component of K+ efflux was not significantly different in SS, SC and CC red cells. The (ouabain + bumetanide)-resistant K+ efflux from SS, SC and CC red cells was stimulated by cell swelling induced by reduction of the osmotic pressure (300 to 220 mosmol/l) and pH (8 to 7) of the flux media (140 mM NaCl). The Cl--dependent K+ efflux stimulated by osmotic swelling highly correlated with the NEM-stimulated component (r = 0.8, p less than 0.001, n = 22) and the acid-pH-induced swelling (r = 0.969, p less than 0.001, n = 22), indicating that it is driven by the K+,Cl- transporter.     

Mammalian NHE2 Na(+)/H+ exchanger mediates efflux of potassium upon heterologous expression in yeast

Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.     

K+ fluxes in Schizosaccharomyces pombe

All living cells accumulate high concentrations of K+ in order to keep themselves alive. To this end they have developed a great diversity of transporters. The internal level of K+ is the result of the net balance between the activities of the K+ influx and the K+ efflux transporters. Potassium fluxes have been extensively studied and characterized in Saccharomyces cerevisiae. However, this is not the case in the fission yeast and, in addition, the information available indicates that both yeasts present substantial and interesting differences. In this paper we have reviewed and summarized the information on K+ fluxes in Schizosaccharomyces pombe. We have included some unpublished results recently obtained in our laboratory and, in particular, we have highlighted the significant differences found between the well-known yeast S. cerevisiae and the fission yeast Sch. pombe.     

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin.

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.     

Amphotericin B-induced changes in K+ content, viability, and ultrastructure of yeast-phase Histoplasma capsulatum.

Yeast-phase cells of Histoplasma capsulatum were challenged with amphotericin B, and membrane perturbation was monitored by K+ efflux. Suspensions of washed cells readily absorbed about 1.12 microgram of amphotericin B per mg (dry weight) and further nonspecific sites were also apparent. The dose-response curve for initial rate of K+ efflux was sigmoidal within the range 0.1 to 1.0 microgram of amphotericin B per ml. A fungistatic concentration of amphotericin B (0.3 microgram/ml) evoked an efflux of 85 to 90% K+ from the cells within 15 min, but cell viability decreased only 13% (yeast phase) or 33% (transformed to mycelial units). Ultrastructural changes in treated cells were detected within 5 min, and the hallmark was expansion of vacuoles during the 1-h monitoring period. In contradistinction to a previous report, the appearance of the protoplasmic membrane was not altered by fungistatic concentration. When treated cells were returned to a fresh growth medium, there was a pronounced lag (20 h). During this apparent recovery phase, the large vacuoles fragmented and returned to normal size. It is proposed that vacuoles of H. capsulatum act as a spatial buffer of considerable survival value to stressed cells.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).