Role of the spleen in the exaggerated polycythemic response to hypoxia in chronic mountain sickness in rats.

In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.

Mathematical analysis of binary activation of a cell cycle kinase which down-regulates its own inhibitor.

In mammalian cells, the heterodimeric kinase cyclin E/CDK2 (EK2) mediates cell cycle progress from G1 phase into S phase. The protein p27Kip1 (p27) binds to and inhibits EK2; but EK2 can phosphorylate p27, and that leads to the deactivation of p27, presumably liberating more EK2 and forming a positive-feedback loop. It has been proposed that this positive-feedback loop gives rise to binary (all-or-none) release of EK2 from its inactive complex with p27. Binary release suggests a bistable biochemical system in which a stable steady state with low EK2 activity is extinguished in a saddle-node bifurcation, causing the system to shift abruptly to a stable steady state with high EK2 activity. Two mathematical models are discussed, one in which free EK2 deactivates p27 in the EK2-p27 inhibitory complex as well as free p27, and one in which the rate of EK2-catalyzed deactivation of free p27 has saturable kinetics with respect to free p27. In general, if inhibitory binding is approximately in equilibrium, bistability requires that there be a potential unstable steady state where the reaction order of p27 deactivation is greater with respect to EK2 than with respect to p27.

Pharmacological tests of the mechanism of the periodic rhythm caused by veratramine in the sinoatrial node of the guinea pig.

1. We investigated the effects of several drugs and extracellular ions on the periodic sinoatrial node rhythm caused by high concentrations of veratramine (>2 microM) in isolated guinea pig sinus atria. 2. During the active phase of this rhythm, pacemaker activity appeared to be due to transient afterdepolarizations resembling the delayed afterdepolarizations attributed to Ca++-induced Ca++ release in cardiac tissue. 3. Ryanodine (200-2200 nM) did not decrease the transient afterdepolarizations, and instead increased the heart rate during the active phase, prolonged the active phase, and sometimes caused conversion to regular rhythm. 4. Dichlorobenzamyl (10-110 microM), a blocker of electrogenic Na+-Ca++ exchange, did not slow or stop beating during periodic rhythm, but rather increased average heart rate and, at a higher concentration, caused conversion to regular rhythm. 5. Ouabain (0.1 microM), an inhibitor of the sodium pump and electrogenic Na+-K+ exchange, had little effect on veratramine periodic rhythm, but at higher concentrations it caused increased average heart rate and conversion to regular rhythm. 6. The chronotropic effect of Ca++ was normally weakly positive; however, in the presence of veratramine, and before the appearance of periodic rhythm, the chronotropic effect of Ca++ was weakly negative, and was associated with destabilization of the heart rate, leading to frequency oscillations or periodic rhythm. 7. Veratramine changed the chronotropic effect of K+ from weakly negative to moderately positive. 8. When half the Na+ or Cl- in the bathing medium was replaced by an impermeant ion, in the absence of veratramine the average heart rate was slightly decreased, whereas, in the presence of veratramine and periodic rhythm the average rate was increased, although the increase was not statistically significant in the case of low Na+. 9. These observations indicate that Ca++-induced Ca++ release, Na+-Ca++ exchange, and probably electrogenic Na+-K+ exchange play no important role in generation of periodic rhythm. The increased K+ dependence suggests an altered pacemaker mechanism.

A simple model of circadian rhythms based on dimerization and proteolysis of PER and TIM.

Many organisms display rhythms of physiology and behavior that are entrained to the 24-h cycle of light and darkness prevailing on Earth. Under constant conditions of illumination and temperature, these internal biological rhythms persist with a period close to 1 day ("circadian"), but it is usually not exactly 24h. Recent discoveries have uncovered stunning similarities among the molecular circuitries of circadian clocks in mice, fruit flies, and bread molds. A consensus picture is coming into focus around two proteins (called PER and TIM in fruit flies), which dimerize and then inhibit transcription of their own genes. Although this picture seems to confirm a venerable model of circadian rhythms based on time-delayed negative feedback, we suggest that just as crucial to the circadian oscillator is a positive feedback loop based on stabilization of PER upon dimerization. These ideas can be expressed in simple mathematical form (phase plane portraits), and the model accounts naturally for several hallmarks of circadian rhythms, including temperature compensation and the per(L) mutant phenotype. In addition, the model suggests how an endogenous circadian oscillator could have evolved from a more primitive, light-activated switch.

Renovascular adaptive changes in chronic hypoxic polycythemia.

BACKGROUND: Chronic hypoxia in rats produces polycythemia, and the plasma fraction falls, reducing renal plasma flow (RPF) relative to renal blood flow (RBF). Polycythemia also causes increased blood viscosity, which tends to reduce RBF and renal oxygen delivery. We studied how renal regulation of electrolyte balance and renal tissue oxygenation (which is crucial for erythropoietin regulation) are maintained in rats during hypoxic exposure. METHODS: Rats of two strains with differing polycythemic responses, with surgically implanted catheters in the urinary bladder, femoral artery, and left renal and right external jugular veins, were exposed to a simulated high altitude (0.5 atm) for 0, 1, 3, 14, and 30 days, after which RPF (para-aminohippurate clearance), glomerular filtration rate (GFR, polyfructosan clearance), hematocrit and blood gases were measured, and RBF, renal vascular resistance and hindrance (resistance/viscosity), renal oxygen delivery, and renal oxygen consumption were calculated. RESULTS: During chronic hypoxia RBF increased, but RPF decreased because of the polycythemia. GFR remained normal because the filtration fraction (FF) increased. Renal vascular resistance decreased, and renal vascular hindrance decreased more markedly. Renal oxygen delivery and consumption both increased. CONCLUSIONS: During chronic hypoxia GFR homeostasis apparently took precedence over RBF autoregulation. The large decrease in renal vascular hindrance suggested that renal vascular remodeling contributes to GFR regulation. The reduced hindrance also prevented a vicious cycle of increasing polycythemia and blood viscosity, decreasing RBF, and increasing renal hypoxia and erythropoietin release.

Studies on the bradycardia and periodic rhythm caused by veratramine in the sinoatrial node of the guinea pig.

In spontaneously beating, isolated guinea pig sinus-atria, veratramine (2.44 microM) slowed the rate of spontaneous depolarization of sinoatrial node cells throughout diastole, markedly slowed the frequency, and often (especially in the presence of high extracellular Ca2+) induced a periodic rhythm. This rhythm consisted of periods of complete inactivity (inactive phases) alternating with periods of apparently normal beating (active phases), with a rising and falling (parabolic) frequency pattern like that of neuronal burst firing. Slight mechanical deformation of the sinoatrial node markedly attenuated the effects of veratramine, and periodic rhythm could not be produced when the sinoatrial node was pinned down for immobilization. During the active phase of periodic rhythm, the rate of spontaneous diastolic depolarization (pacemaker potential) in pacemaker cells rose and fell with the frequency, while contrariwise, the maximum rate of depolarization fell and then rose. The last beat in the active phase was followed by a small transient afterdepolarization, which had the appearance of an abortive pacemaker potential that failed to reach threshold for triggering an action potential. The effects of various programs of electrical stimulation and pacing indicated that activity of the sinoatrial node, whether spontaneous or driven, has two effects on the amplitude of afterdepolarization, a short-lasting cumulative facilitory effect and a long-lasting cumulative inhibitory effect. Veratramine periodic rhythm arises from the interplay of these two effects, with abrupt cessation of beating whenever the afterdepolarization amplitude falls below the threshold for triggering an action potential. It is suggested that the inhibitory effect may be due to inactivation of the slow inward current and the facilitory effect may be due to one or more of the depolarizing currents activated by intracellular Ca2+.

Bistable biochemical switching and the control of the events of the cell cycle.

Some of the events of the cell cycle appear to be triggered by a bistable mechanism. A bistable biochemical system can respond to a small, slow signal and is carried by positive feedback from one stable steady state directly to another, in an all-or-none manner. Slow or subthreshold stimuli do not cause accommodation or loss of excitability. Switching is not readily reversible by removing the stimulus, i.e. there is hysteresis: reversal generally requires a stronger, opposite stimulus. Biochemically, bistable biochemical switching requires positive feedback, and mechanisms for stabilizing the system against premature activation and for destabilization in response to a biological signal. Three bistable biochemical models, all suggested by reported experimental observations, are described and analysed. These models suggest that a titratable inhibitor may play an important part in bistable switching, because the end-point of titration can form a natural threshold for enhancement of positive feedback.

Chemical kinetic theory: understanding cell-cycle regulation.

Progress of a cell through its reproductive cycle of DNA synthesis and division is governed by a complex network of biochemical reactions controlling the activities of both M-phase- and S-phase-promoting factors. Standard chemical kinetic theory provides a disciplined method for expressing the molecular biologists' diagrams and intuition in precise mathematical form, so that qualitative and quantitative implications of our 'working models' can be derived and compared with experiment.

A model for a bistable biochemical trigger of mitosis.

The activation of maturation promoting factor (MPF, cyclin B/Cdc2), which starts mitosis, is modeled as a bistable biochemical switch or trigger. A small, slow parameter change can cause an abrupt transition by a saddle-node bifurcation from a stable steady state of low activity to one of high activity. The switch is not reversed if the parameter change is reversed (hysteresis). The dynamical features necessary for this triggering action are the presence of two stable steady states (low-activity and high-activity), and one unstable steady state. The key biochemical kinetic features of the model are (1) mutual activation by MPF and Cdc25, which makes the activation of MPF effectively autocatalytic, and (2) binding of MPF by Suc1, which inhibits MPF autocatalysis and stabilizes the low-activity steady state until the amount of MPF begins to approach or exceed stoichiometrically the amount of Suc1, then allows strong autocatalysis and full activation. The special virtues of bistable triggering, and the general types of biochemical mechanism which can produce it, are discussed.

Theoretical dynamics of the cyclin B-MPF system: a possible role for p13suc1.

In dividing cells, entry into mitosis is caused by maturation promoting factor (MPF), which is formed autocatalytically by activation of a complex of p34cdc2 and cyclin B. This biochemical system may oscillate, causing repeated mitosis. It is shown mathematically that the oscillatory tendency would be enhanced by a cofactor which binds to MPF and inhibits its autocatalytic action. A candidate for such a cofactor is the suc1 gene product p13, which binds to p34cdc2/cyclin B complex and inhibits MPF-induced MPF activation. At a steady rate of cyclin biosynthesis, with small amounts converted to MPF, p13suc1 would have to be titrated by MPF before autocatalysis could begin. This would have three possibly important effects: (1) it would determine the 'threshold' cyclin accumulation (and hence the corresponding time-delay) for MPF activation; (2) it would cause the accumulation of a backlog of MPF precursor (tyrosine-phosphorylated p34cdc2/cyclin B) sufficient to produce a substantial MPF pulse when MPF autocatalysis begins; (3) it would give the autocatalysis a high reaction order, which tends to destabilize the steady state, promote autonomous oscillations, and enhance the triggering property (excitability) of the system. The MPF pulse generated by this system may be essential for the proper triggering of the events of M phase, including the cyclin degradation which inactivates MPF at the end of M phase. This model offers explanations for several puzzling effects of p13suc1, including the fact that p13suc1, though an inhibitor of MPF activation, is nevertheless necessary for mitosis.

Possible role of pulmonary blood volume in chronic hypoxic pulmonary hypertension.

Chronic hypoxia increases the total blood volume (TBV) and pulmonary arterial blood pressure (Ppa) and induces pulmonary vascular remodeling. The present study was undertaken to assess how the pulmonary blood volume (PBV) changes during hypoxia and the possible role of PBV in chronic hypoxic pulmonary hypertension. A novel method has been developed to measure the TBV, PBV, and Ppa in conscious rats. The method consists of chronic implantation of a loose ligature around the ascending aorta and pulmonary artery, so that when the ligature is drawn tightly, it traps the blood in the pulmonary vessels and left heart and simultaneously kills the rat. The pulmonary veins are then ligated to separate the left ventricular blood volume from the PBV. This surgical approach, together with chronic catheterization of the pulmonary artery and the use of 51Cr-labeled red blood cells, allows measurement of TBV, PBV, and Ppa. This method has been used to analyze the relationships between TBV and PBV and between Ppa or right ventricular hypertrophy and PBV in two rat strains with markedly different TBV and Ppa responses to chronic hypoxia. PBV per given lung weight did not increase and even decreased during hypoxia despite marked increases in TBV. There was a close correlation between Ppa or right ventricular hypertrophy and PBV in the two strains of chronically hypoxic animals, suggesting that a greater PBV plays a significant role in the development of severe chronic hypoxic pulmonary hypertension in the altitude-susceptible Hilltop rats.

Does atrial natriuretic factor protect against right ventricular overload? I. Hemodynamic study.

We studied the effects of synthetic atrial natriuretic factor (ANF, 28-amino acid peptide) on base-line perfusion pressures and pressor responses to hypoxia and angiotensin II (ANG II) in isolated rat lungs and on the following hemodynamic and renal parameters in awake, chronically instrumented rats: cardiac output (CO), systemic (Rsa) and pulmonary (Rpa) vascular resistances, ANG II- and hypoxia (10.5% O2)-induced changes in Rsa and Rpa, and urine output. Intra-arterial ANF injections lowered base-line perfusion pressures and blunted hypoxia- and ANG II-induced pressor responses in the isolated lungs. Bolus intravenous injection of ANF (10 micrograms/kg) into intact rats decreased CO and arterial blood pressures of both systemic and pulmonary circulations and increased Rsa. ANG II (0.4 micrograms/kg) increased both Rsa and Rpa, and hypoxia increased Rpa alone in the intact rats. ANF (10 micrograms/kg) inhibited both ANG II- and hypoxia-induced increases in Rpa but did not significantly affect the ANG II-induced increase in Rsa. The antagonistic effect of ANF on pulmonary vasoconstriction was reversible and dose-dependent. The threshold doses of ANF required to inhibit pulmonary vasoconstriction were in the same range as those required to elicit diuresis and natriuresis. The data demonstrate that ANF has a preferential relaxant effect on pulmonary vessels constricted by hypoxia or ANG II. Both the renal and the pulmonary vascular effects of ANF may represent fundamental physiological actions of ANF. These actions may serve as a negative feedback control system that protects the right ventricle from excessive mechanical loads.

Cyanide release from nitroprusside in the isolated, perfused, bloodless liver and hindlimbs of the rat.

When sodium nitroprusside in artificial medium was perfused through the isolated liver and hindlimbs of a rat at the near physiological flow rate of 8.5 ml min-1, free cyanide was found in the perfusate. The liver reached a steady-state ratio of cyanide released/nitroprusside perfused of about 1.5 (or approximately 30% of the total nitroprusside cyanide) within 15 min, and maintained that rate for about 1.5 hr. In the hindlimbs cyanide was released at a much slower rate (7.5 to 18.8% of the total), and the release did not achieve a steady state even after 1.5 hr. Even after small corrections for cyanide extraction by both tissues, the rate of cyanide release by either tissue was probably more rapid than that resulting from static incubations in blood.