Effects of beta-adrenergic catecholamines on potassium transport in turkey erythrocytes.

Isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a 2- to 4-fold increase in bidirectional potassium fluxes in turkey erythrocytes. Ouabain, which does not alter catecholamine-stimulated cellular cyclic AMP, inhibits potassium influx by 50 to 70%, does not alter potassium outflux or isoproterenol-stimulated potassium influx, but increases isoproterenol-stimulated potassium outflux. Stimulation of potassium transport by isoproterenol can be reproduced by adding cyclic AMP to the medium and is inhibited by propranolol or dichloroisoproterenol but not by phentolamine. Theophylline at concentrations which inhibit cyclic nucleotide phosphodiesterase in isolated turkey erythrocyte plasma membranes by greater than 90%, does not augment isoproterenol stimulation of cellular cyclic AMP or of potassium transport but does potentiate stimulation of potassium influx produced by adding cyclic AMP to the medium. Isoproterenol-stimulated cellular cyclic AMP increases steadily for at least 2 hours. Potassium transport, however, increases rapidly, becomes maximal after 20 to 30 min of incubation, and thereafter decreases progressively so that after 2 hours of incubation potassium fluxes are only slightly greater than for the control. Ouabain prolongs the duration of catecholamine-stimulated potassium influx and potassium outflux, reflecting its ability to relieve the refractoriness developed by turkey erythroyctes to endogenous cyclic AMP.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.     

Development of hormonal regulation of ion transport in embryonic chick red cells

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8- or 9-day embryos are stimulated little or not at all. The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport. The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na-K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na-K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain-sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.     

Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells.

The COOH-terminal octapeptide of cholecystokinin (CCK-OP) and carbamylcholine each increased calcium outflux, cellular cyclic GMP and amylase secretion in dispersed guinea pig pancreatic acinar cells. Following addition of CCK-OP or carbamylcholine, cellular cyclic GMP increased as early as 15 s, became maximal after 1 to 2 min, and then decreased steadily during the subsequent incubation. For both CCK-OP and carbamylcholine there was close agreement between the dose-response curve for stimulation of calcium outflux and that for increase of cellular cyclic GMP. With CCK-OP an effect on both functions could be detected at 10(-10) M and maximal stimulation occurred at 3 X 10(-8) M. With carbamylcholine an effect on both functions could be detected at 10(-5) M and maximal stimulation occurred at 3 X 10(-3) M. Atropine inhibited stimulation of both cyclic GMP and calcium outflux by carbamylcholine but not by CCK-OP. Stimulation of calcium outflux or cellular cyclic GMP by CCK-OP or carbamylcholine did not require extracellular calcium since stimulation occurred in a calcium-free, ethylene glycol bis(beta, beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-containing solution. The divalent cation ionophore A-23187 increased bidirectional fluxes of calcium, cellular cyclic GMP and secretion of amylase from dispersed pancreatic acinar cells. Like CCK-OP and carbamylcholine, the ionophore stimulated calcium outflux and cellular cyclic GMP in a calcium-free, EGTA-containing solution. These results suggest that in pancreatic acinar cells the initial step in the sequence of events mediating the action of ionophore as well as that of CCK-OP and carbamylcholine is stimulation of calcium outflux, and that this stimulation then increases cellular cyclic GMP.     

Effects of ATP and cyclic AMP on the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes

As turkey erythrocytes were progressively depleted of ATP by preincubation with dinitrophenol, the (Na+ + K+ + 2Cl-)-cotransport system (assayed by the bumetanide-sensitive fraction of 86Rb+ influx) became less responsive to activation. The dependence upon intracellular ATP concentration was significantly steeper for transport activated by hypertonic shock (halfmaximal activity at 0.7 mM ATP) than for that activated by either epinephrine or cyclic AMP (halfmaximal activity at 1.7 mM ATP). Upon removal of epinephrine or cyclic AMP from cells that had been preincubated with those substances, bumetanide-sensitive transport activity declined sharply, even though the intracellular cyclic AMP concentration was still over 10-fold that required to maximally activate the transport system. These data are in agreement with the notion that the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by cyclic AMP, presumably through the 'classical' pathway involving a protein kinase. They do however indicate that some other, as yet undefined aspect of cyclic AMP metabolism is important for the maintenance of transport activity.     

Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.     

Further studies of hormone-sensitive sodium and potassium transport in red cells from developing chick embryos

Sodium and potassium transport in the definitive series of chick embryo red cells changes significantly, both qualitatively and quantitatively, during maturation. Sodium efflux and potassium influx consist of three parts: a ouabain-sensitive, a furosemide-sensitive, and a ouabain-furosemide-insensitive component. In chick red cells of most ages, the ouabain-sensitive and furosemide-sensitive parts of the cation fluxes do not overlap. Cation transport in the more mature red cells is increased significantly by epinephrine, whereas cation transport in red cells from younger embryos is stimulated much less. This is a beta-adrenergic effect of epinephrine and is mediated by cyclic AMP. The relative lack of response in younger embryos is not due to the absence of beta-adrenergic receptor or the lack of production of cyclic AMP. Ouabain has no effect on the hormone-sensitive sodium or potassium transport. On the other hand, furosemide nearly completely abolishes the effect of epinephrine. In addition, there is a good correlation between furosemide-sensitive components of both sodium and potassium transport and the epinephrine-sensitive component. Furosemide has no effect on cyclic AMP levels in the presence or absence of epinephrine. This suggests that furosemide may act directly on the cation transport system. In the red cells from younger embryos, furosemide-sensitive units are present but cannot be fully activated by epinephrine. Therefore, the lack of the hormone effect on cation movements in these cells is consistent with the view that the appropriate units are present, but do not respond fully to intracellular cyclic AMP levels.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Mechanisms altered beta-adrenergic responsiveness in the hyperthyroid and hypothyroid turkey erythrocyte

Studies on the relationship between thyroid hormone and the beta-adrenergic catecholamines have been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associated biochemical and physiological indices of beta receptor function, including agonist stimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulation of potassium ion influx. Erythrocytes obtained from hypothyroid turkeys showed a marked (approximately 50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMP levels were similarly reduced. The sensitivity of these cells to agonist-stimulated potassium influx was significantly decreased, but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relationship between beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy, the level of agonist-stimulated potassium influx is identical in hypothyroid and normal erythrocytes, and that the diminished physiological sensitivity of the hypothyroid cell is attributable in its entirety to a reduction in beta receptor number per se. The results obtained in the hyperthyroid turkey erythrocyte were strikingly different. Here, beta receptor number, binding affinity for agonists and antagonists, catecholamine-sensitive adenylate cyclase activity, and maximal cyclic AMP levels were all unchanged. In contrast, maximal agonist-stimulated potassium ion transport was markedly reduced, while the concentration of isoproterenol required for half-maximal stimulation was only slightly increased. Analysis of the relationship between beta receptor number, agonist concentration, and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy, the stimulation of monovalent cation influx is markedly blunted in the hyperthyroid cell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivity to catecholamines is directly attributable to a reduction in beta receptor number, the primary abnormality responsible for diminished catecholamine responsiveness in the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte. Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K+ concentration on both [3H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between beta-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [3H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K+ concentrations, and at each K+ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K+ concentrations. In contrast to basal potassium influx, which is 50-70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K+, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to beta-adrenergic receptor occupany over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy beta-adrenergic receptor sites function independently of each other. Analysis of the relation of catecholamine-dependent potassium transport to the number of beta-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

Ouabain-Insensitive Sodium Movements in the Human Red Blood Cell

Red blood cells exposed to ouabain are capable of net Na outflux against an electrochemical gradient; the net outflux is inhibited by the diuretic, furosemide. In ouabain-treated cells, both the unidirectional Na outflux and the unidirectional Na influx are inhibited by furosemide. Furosemide also inhibits the ouabain-sensitive Na-Na exchange accomplished by the Na-K pump in K-free solutions. From the interaction of extracellular K, furosemide, and ouabain with the transport system, it seems possible that the ouabain-insensitive Na outflux is accomplished by the same mechanism that is responsible for the ouabain-sensitive Na-K exchange. The ouabain-insensitive Na outflux is increased by extracellular Na, and the influx increases as the intracellular Na increases. In fresh cells, high extracellular K concentrations decrease the ouabain-insensitive Na outflux and increase the ouabain-insensitive Na influx. When the rate constant for sodium outflux and the rate constant for sodium influx in ouabain-treated cells are plotted against the extracellular K concentration, the curves obtained are mirror images of each other. In starved cells, extracellular K increases the ouabain-insensitive Na outflux as does extracellular Na, and it has little effect on the Na influx.     

Depolarization-evoked accumulation of cyclic AMP in brain slices: inhibition by exogenous adenosine deaminase

In guinea pig cerebral cortical slices labeled during a prior incubation with radioactive adenine, electrical stimulation or the presence of depolarizing agents such as veratridine, ouabain, and high concentrations of K⁺ elicit a marked accumulation of radioactive cyclic AMP. This accumulation is reduced in all cases by the presence of theophylline, a compound that antagonizes the stimulatory effects of adenosine on cyclic AMP accumulation in brain slices. Exogenous adenosine deaminase also reduced the accumulation of cyclic AMP elicited by electrical stimulation, veratridine, and high concentrations of K⁺. Thus, adenosine formed in neuronal compartments under depolarizing conditions appears to be released into the extracellular medium as a prerequisite to stimulation of the cyclic AMP-generating system. Adenosine deaminase does not prevent the reduction in levels of ATP under depolarizing conditions, nor does it antagonize the accumulation of cyclic AMP elicited by a combination and norepinephrine. Adenosine deaminase does not, however, prevent the accumulations of cyclic AMP elicited by the depolarizing agent, ouabain.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte: Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K⁺ concentration on both [³H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between β-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [³H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K⁺ concentrations, and at each K⁺ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K⁺ concentrations. In contrast to basal potassium influx, which is 50–70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K⁺, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to β-adrenergic receptor occupancy over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy β-adrenergic receptor sites function independently of each other.Analysis of the relation of catecholamine-dependent potassium transport to the number of β-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

Activation of sodium transport in human erythrocytes by beta-adrenoceptor stimulation in vivo

Beta-adrenoceptor stimulation in vivo shifts potassium into the cells. To examine whether human erythrocytes participate in this process, we measured, along with serum or plasma potassium, the concentrations of potassium and sodium in erythrocytes. Beta-adrenoceptor stimulation was obtained by infusion of either fenoterol or hexoprenaline into 6 volunteers at rest or by endogenous amines provoked in 14 volunteers during ergometric exercise. Metabolic effects were followed at rest on serum insulin, C-peptide, and growth hormone levels, and during exercise on pH on lactate concentration in blood. The potassium concentration (mean +/- S.E.M.) dropped (p less than 0.01) in serum from 4.64 +/- 0.37 to 3.19 +/- 0.43 mmol x l-1 in the first hour at rest and in plasma from 5.70 +/- 0.93 to 4.63 +/- 0.45 in 90 sec directly after exercise. The concentration of erythrocyte sodium dropped (p less than 0.001) from 9.68 +/- 0.73 to 8.81 +/- 0.62 mmol x l-1 in cells and from 9.62 +/- 1.16 to 8.55 +/- 1.24 during exercise for 90 s, respectively. Changes in the concentration ratio of cellular sodium to potassium confirmed this sodium shift. An increased sodium transport in erythrocytes due to beta-adrenoceptor stimulation in vivo appears to complement a shift of serum potassium into the cells and may be mediated by the membrane-bound sodium, potassium ATPase.     

Stimulation of alanine transport and metabolism by dibutyryl cyclic AMP in the hepatocytes from fed rats. Assessment of transport as a potential rate-limiting step for alanine metabolism

(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2-5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na+ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na+ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

A requirement of bicarbonate for Ca2+-induced elevations of cyclic AMP in guinea pig spermatozoa

Ca2+ causes less than 2-fold elevations of guinea pig sperm cyclic AMP concentrations when cells are incubated in a minimal culture medium in the absence of bicarbonate (HCO3-). However, in the presence of HCO3-, Ca2+ increases cyclic AMP by as much as 25-fold within 1 min. The (Ca2+, HCO3-)-induced elevations occur in either the presence or absence of the permeant anions, pyruvate and lactate. In the absence of extracellular Ca2+, HCO3- elevates cyclic AMP only slightly. The effect of HCO3- is concentration-dependent, with maximal responses obtained at concentrations of greater than 25 mM. Ca2+ (25 mM HCO3-) at concentrations of less than 100 microM causes one-half-maximal elevations of cyclic AMP. The (Ca2+, HCO3-)-induced elevations of cyclic AMP are observed at various extracellular pH values (7.5-8.5) and in the presence or absence of extracellular Na+ or K+. NH4Cl does not elevate sperm cyclic AMP concentrations and does not greatly alter the (Ca2+, HCO3-)-induced elevations. the putative Ca2+ transport antagonist, D-600 (100 microM), completely blocks the (Ca2+, HCO3-)-induced elevations of cyclic AMP. A23187, in the presence but not in the absence of extracellular Ca2+, increases sperm cyclic AMP but does not further elevate cyclic AMP in HCO3(-)-treated cells. These studies establish that Ca2+-dependent elevations of cyclic AMp in guinea pig spermatozoa are dependent on the presence of HCO3- and suggest that HCO3- is required for the uptake (exchange) or membrane sequestration of small amounts of physiologically active Ca2+.