大鼠延髓外周橄榄腹外侧核在中枢化学感受中的作用

本工作采用微量注射、电损毁、电刺激和微电泳的方法,探讨了大鼠延髓外周橄榄腹外侧核（ＬＶＰＯ）的否真正参与中枢化学感受功能。实验在３８只雄性ＳＤ大鼠上进行。结果表明：（１）微量注射酸化人工脑脊液于ＬＶＰＯ可引起隔神经放电活动明显加强。（２）微电泳给予Ｈ＾＋对ＬＶＰＯ的自发放电单位主要引起兴奋反应,微电泳给予Ｈ＾＋引起兴奋反应的部分单位也可被腹外侧表面微量注射酸化人工ＣＳＦ所兴奋。（３）损毁ＬＶＰＯ后     

Differential respiratory effects of HCO3- and CO2 applied on ventral medullary surface of rats

To estimate whether H+ is the unique stimulus of the medullary chemosensor, ventilatory effects of HCO3- and/or CO2 applied on the ventral medullary surface using an improved superfusion technique and of CO2 inhalation were compared in halothane-anesthetized spontaneously breathing rats. Superfusion with low [HCO3-]-acid mock cerebrospinal fluid (CSF) (normal Pco2) induced a significant increase in ventilation, with an accompanying reduction in endtidal Pco2 (PETco2). High [HCO3-]-alkaline CSF depressed ventilation. Changes in Pco2 of superfusing CSF, on the other hand, had no significant effect despite the similar changes in pH. Simultaneous decrease in [HCO3-] and Pco2 of mock CSF with normal pH also maintained stimulated respiration. CO2 inhalation during superfusion with various [HCO3-] solutions caused further increase in ventilation as PETco2 increased. The results suggest that the surface area of the rat ventral medulla contains HCO3- (or H+)-sensitive respiratory neural substrates which are, however, little affected by CO2 in the subarachnoid fluid. A CO2 (or CO2-induced H+)-sensitive chemosensor responsible for the increase in ventilation during CO2 inhalation may exist elsewhere functionally apart from the HCO3- (or H+)-sensitive sensor in the examined surface area.     

Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO2.

The exact location of the central respiratory chemoreceptors sensitive to changes in PCO2 has not yet been determined. To avoid the confounding effects of the cerebral circulation, we used the in vitro brain stem-spinal cord of neonatal rats (1-5 days old) to identify areas within 500 microns of the ventral surface of the medulla where changes in PCO2 evoked a sudden increase in the rate of respiratory neural activity. The preparation was superfused with mock cerebrospinal fluid (CSF) while maintained at constant temperature (26 +/- 1 degrees C) and pH (7.34). Respiratory frequency increased linearly with decreases in superfusate pH (r2 = 0.92, P less than 0.001), indicating that the respiratory circuitry for the detection of CO2 and stimulation of breathing was intact in this preparation. The search for central chemoreceptors was performed with a specially designed micropipette that allowed microejection of 2-10 nl of mock CSF equilibrated with different CO2-O2 gas mixtures. The pipette was advanced in 50- to 100-microns steps by use of a microdrive to a maximum depth of 500 microns from the surface of the ventral medulla. Depending on the location of the micropipette, ejection of CO2-acidified mock CSF at depths of 100-350 microns below the ventral surface of the medulla stimulated neural respiratory output. Using this response as an indication of the location of central respiratory chemoreceptors, we found that chemoreceptive elements were located in a column in the ventromedial medulla extending from the hypoglossal rootlets caudally to an area 0.75 mm caudal to VI nerve in the rostral medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between cerebro-spinal fluid pH and pulmonary ventilation of the South American lungfish, Lepidosiren paradoxa (Fitz.)

The respiratory control in land vertebrates (Tetrapoda) is mainly linked to regulation of acid-base status, which involves peripheral and central chemoreceptors. The lungfish (Dipnoi) might constitute the sister group of all land vertebrates (Tetrapoda) and possess a combination of real lungs and reduced gills. In this context, we evaluated the possible presence of central respiratory chemoreceptors in the South American Lungfish, Lepidosiren paradoxa. Pulmonary ventilation and respiratory frequency increased significantly with reductions of CSF pH by means of mock CSF solutions. This suggests that Lepidosiren possess central acid-base receptors.     

CO2/H(+) sensing: peripheral and central chemoreception

H(+) is maintained constant in the internal environment at a given body temperature independent of external environment according to Bernard's principle of "milieu interieur". But CO2 relates to ventilation and H(+) to kidney. Hence, the title of the chapter. In order to do this, sensors for H(+) in the internal environment are needed. The sensor-receptor is CO2/H(+) sensing. The sensor-receptor is coupled to integrate and to maintain the body's chemical environment at equilibrium. This chapter dwells on this theme of constancy of H(+) of the blood and of the other internal environments. [H(+)] is regulated jointly by respiratory and renal systems. The respiratory response to [H(+)] originates from the activities of two groups of chemoreceptors in two separate body fluid compartments: (A) carotid and aortic bodies which sense arterial P(O2) and H(+); and (B) the medullary H(+) receptors on the ventrolateral medulla of the central nervous system (CNS). The arterial chemoreceptors function to maintain arterial P(O2) and H(+) constant, and medullary H(+) receptors to maintain H(+) of the brain fluid constant. Any acute change of H(+) in these compartments is taken care of almost instantly by pulmonary ventilation, and slowly by the kidney. This general theme is considered in Section 1. The general principles involving cellular CO2 reactions mediated by carbonic anhydrase (CA), transport of CO2 and H(+) are described in Section 2. Since the rest of the chapter is dependent on these key mechanisms, they are given in detail, including the role of Jacobs-Stewart Cycle and its interaction with carbonic anhydrase. Also, this section deals briefly with the mechanisms of membrane depolarization of the chemoreceptor cells because this is one mechanism on which the responses depend. The metabolic impact of endogenous CO2 appears in the section with a historical twist, in the context of acclimatization to high altitude (Section 3). Because low P(O2) at high altitude stimulates the peripheral chemoreceptors (PC) increasing ventilation, the endogenous CO2 is blown off, making the internal milieu alkaline. With acclimatization however ventilation increases. This alkalinity is compensated in the course of time by the kidney and the acidity tends to be restored, but the acidification is not great enough to increase ventilation further. The question is what drives ventilation during acclimatization when the central pH is alkaline? The peripheral chemoreceptor came to the rescue. Its sensitivity to P(O2) is increased which continues to drive ventilation further during acclimatization at high altitude even when pH is alkaline. This link of CO2 through the O2 chemoreceptor is described in Section 4 which led to hypoxia-inducible factor (HIF-1). HIF-1 is stabilized during hypoxia, including the carotid body (CB) and brain cells, the seat of CO2 chemoreception. The cells are always hypoxic even at sea level. But how CO2 can affect the HIF-1 in the brain is considered in this section. CO2 sensing in the central chemoreceptors (CC) is given in Section 5. CO(2)/H(+) is sensed by the various structures in the central nervous system but its respiratory and cardiovascular responses are restricted only to some areas. How the membranes are depolarized by CO2 or how it works through Na(+)/Ca(2+) exchange are discussed in this section. It is obvious, however, that CO2 is not maintained constant, decreasing with altitude as alveolar P(O2) decreases and ventilation increases. Rather, it is the [H(+)] that the organism strives to maintain at the expense of CO2. But then again, [H(+)] where? Perhaps it is in the intracellular environment. Gap junctions in the carotid body and in the brain are ubiquitous. What functions they perform have been considered in Section 6. CO2 changes take place in lung alveoli where inspired air mixes with the CO2 from the returning venous blood. It is the interface between the inspired and expired air in the lungs where CO2 change is most dramatic. As a result, various investigators have looked for CO2 receptors in the lung, but none have been found in the mammals. Instead, CO2/H(+) receptors were found in birds and amphibians. However, they are inhibited by increasing CO2/H(+), instead of stimulated. But the afferent impulses transmitted to the brain produced stimulation in the efferents. This reversal of afferent-efferent inputs is a curious situation in nature, and this is considered in Section 7. The NO and CO effects on CO2 sensing are interesting and have been briefly mentioned in Section 8. A model for CO2/H(+) sensing by cells, neurons and bare nerve endings are also considered. These NO effects, models for CO2/H(+) and O2-sensitive cells in the CNS have been considered in the perspectives. Finally, in conclusion, the general theme of constancy of internal environment for CO2/H(+) is reiterated, and for that CO2/H(+) sensors-receptors systems are essential. Since CO2/H(+) sensing as such has not been reviewed before, the recent findings in addition to defining basic CO2/H(+) reactions in the cells have been briefly summarized.     

Intracisternal DIDS enhances ventilatory response to rebreathing CO2 in rats

In 11 anesthetized rats, we tested the hypothesis that carrier-mediated anion transport in part determines the medullary chemoreceptor response to acute hypercapnia by infusing the transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in mock cerebrospinal fluid (CSF) into the cisterna magna. In five additional rats with sham CSF infusion, we found no effect of mock CSF on the response to rebreathing CO2. Dye infused into the cistern stained the putative chemoreceptor areas on the ventral surface of the medulla. DIDS, at 10 to 1,000 nM, increased the respiratory response to CO2 in a dose-related manner but had no effect on arterial pressure or heart rate. At 1,000 nM, the hypercapnic minute ventilation response was almost doubled because of both volume and rate of breathing. We conclude that the net effect of anion transport is to mitigate the stimulus to the medullary chemoreceptors during acute hypercapnia.     

Effects of altered CSF [H+] on ventilatory responses to exercise in the awake goat

We investigated the effects of selective large changes in the acid-base environment of medullary chemoreceptors on the control of exercise hyperpnea in unanesthetized goats. Four intact and two carotid body-denervated goats underwent cisternal perfusion with mock cerebrospinal fluid (CSF) of markedly varying [HCO-3] (CSF [H+] = 21-95 neq/l; pH 7.68-7.02) until a new steady state of alveolar hypo- or hyperventilation was reached [arterial PCO2 (PaCO2) = 31-54 Torr]. Perfusion continued as the goats completed two levels of steady-state treadmill walking [2 to 4-fold increase in CO2 production (VCO2)]. With normal acid-base status in CSF, goats usually hyperventilated slightly from rest through exercise (-3 Torr PaCO2, rest to VCO2 = 1.1 l/min). Changing CSF perfusate [H+] changed the level of resting PaCO2 (+6 and -4 Torr), but with few exceptions, the regulation of PaCO2 during exercise (delta PaCO2/delta VCO2) remained similar regardless of the new ventilatory steady state imposed by changing CSF [H+]. Thus the gain (slope) of the ventilatory response to exercise (ratio of change in alveolar ventilation to change in VCO2) must have increased approximately 15% with decreased resting PaCO2 (acidic CSF) and decreased approximately 9% with increased resting PaCO2 (alkaline CSF). A similar effect of CSF [H+] on resting PaCO2 and on delta PaCO2/VCO2 during exercise also occurred in two carotid body-denervated goats. Our results show that alteration of the gain of the ventilatory response to exercise occurs on acute alterations in resting PaCO2 set point (via changing CSF [H+]) and that the primary stimuli to exercise hyperpnea can operate independently of central or peripheral chemoreception.     

Recombinant hTASK1 is an O(2)-sensitive K(+) channel

Hypoxic inhibition of background K(+) channels is crucial to O(2) sensing by chemoreceptor tissues, but direct demonstration of O(2) sensitivity by any member of this K(+) channel family is lacking. HEK293 cells were transfected with a pcDNA3.1-hTASK1 construct; expression of hTASK1 was verified using RT-PCR and immunocytochemistry. Whole-cell K(+) currents of cells stably expressing hTASK-1 were, as anticipated, extremely sensitive to extracellular pH, within the physiological range (IC(50) approximately 7.0). All cells expressing this signature pH sensitivity were acutely modulated by pO(2); reduction of pO(2) from 150 to <40 mmHg (at pH 7.4) caused rapid and reversible suppression of pH-sensitive K(+) currents. Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O(2)-sensitive current was dependent on the extracellular pH. These data represent the first direct verification that hTASK1 is O(2)-sensitive and reinforce the idea that this K(+) channel is key to O(2) sensing in chemoreceptors.     

Central cardiorespiratory effects of glutamate in dogs

Metabolism of certain amino acid neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA) are closely linked in the brain to CO2 fixation and H+ metabolism. Additionally they may also affect central modulation of cardiorespiratory function. Therefore central cardiorespiratory effects of L-glutamate were determined in lightly anesthetized dogs using ventriculocisternal perfusion with artificial cerebrospinal fluid (CSF) (pH 7.25-7.28) containing 30 or 60 mM glutamate at a flow rate of 1.0 ml/min for 20 min followed by perfusion with artificial CSF alone. Tidal volume and minute ventilation increased with 60 mM glutamate, as did respiratory drive. These changes returned to normal with mock CSF perfusion. Glutamate (30 mM) had no significant effect on ventilation. At both concentrations, glutamate significantly increased mean femoral arterial pressure and mean pulmonary arterial pressure, which was accompanied by bradycardia. All these increases rapidly returned to normal with mock CSF perfusion. Cardiac output and pulmonary capillary wedge pressure did not change with glutamate perfusion. The results suggest that glutamate may have a significant central excitatory role in modulation of ventilatory drive as well as of hemodynamic functions.     

Central chemoreceptors

When all peripheral chemoreceptors are denervated, animals continue to show increased ventilation when made to breathe CO2, indicating that receptors within the brain ("central chemoreceptors") are excited by acidity or changes in CO2. No cells have been identified within the brain that are indisputedly chemoreceptors for CO2 or H+, but there is abundant evidence that respiration can be affected by chemical, electrical, and thermal stimuli applied locally to the ventral surface of the medulla. Furthermore, the actions of traditional central chemical respiratory stimuli can be blunted or abolished after inhibition of neural function within this ventrolateral medullary shell (VMS). The VMS is an integrative region for cardiovascular and respiratory function and may be involved in nociception. The distinction between the former two is not always clear, but recent studies using microinjection techniques seem promising for identifying the respiratory substrates. The many recent advances elucidating anatomic connections between the VMS and other brain regions are important but do not directly address the question of the site of central respiratory chemosensitivity. Knowledge of such connections, however, should provide more definitive opportunities for addressing this question.     

Digital Computer Simulation of Respiratory Response to Cerebrospinal Fluid PCO2 in the Cat

The respiratory control system is treated as linear with a transmission delay between ventilation and sensing points (chemoreceptors). To the accepted variables involving body gas stores, ventilatory effects, transmission effects, and steady state pH, P(CO2), P(O2) chemoreceptor response, certain detailed analysis of the central receptors have been added. By construction of a model for medullary CO(2) receptor utilizing expected values of CNS (central nervous system) circulation, CO(2) production, and tissue-buffering effects, results of experimental observation of the effects of alteration of CSF were simulated. The inclusion of CSF effects also allowed simulation of the response to alteration in inspired CO(2), hyperventilation, and the periodic breathing with prolongation of circulation time.     

Anatomical arrangement of hypercapnia-activated cells in the superficial ventral medulla of rats.

The anatomical structure of central respiratory chemoreceptors in the superficial ventral medulla of rats was studied by using hypercapnia-induced c-fos labeling to identify cells directly stimulated by extracellular pH or PCO(2). The distribution of c-fos-positive cells was found to be predominantly perivascular to surface vessels. In the superficial ventral medullary midline, parapyramidal, and ventrolateral regions where c-fos-positive cells were concentrated, we found a common, characteristic, anatomical architecture. The medullary surface showed an indentation covered by a surface vessel, and the marginal glial layer was thickened. We classified c-fos-positive cells into two types. One (type I cell) was small, was located inside the marginal glial layer and close to the medullary surface, and surrounded fine vessels. The other (type II cell) was large and located dorsal to the marginal glial layer. c-fos Expression under synaptic blockade suggested that type I cells are intrinsically chemosensitive. The chemosensitivity of surface cells (possible type I cells) surrounding vessels was confirmed electrophysiologically in slice preparations. We suggest that this characteristic anatomical structure may be the central chemoreceptor.     

Expression of Nox1 in 3T3 cells increases cellular acid production but not proton conductance

The role of the NADPH oxidase homolog 1 (Nox1) in plasma membrane H(+) conductance and cellular H(+) production was investigated in 3T3 cells stably expressing Nox1 (Nox1 3T3) compared to vector-expressing control cells (mock 3T3). In whole cell patch clamp experiments both Nox1 and mock 3T3 expressed a similar H(+) conductance (Nox1 3T3, 13.2+/-8.6 pS/pF; mock 3T3, 16.6+/-13.4 pS/pF) with a number of similar characteristics (e.g., current-voltage relations, current activation kinetics, Zn(2+)-sensitivity). When the intracellular pH of cells was alkalinized with NH(4)Cl, rates of intracellular acidification were significantly higher in Nox1 3T3 compared to mock 3T3. Nox1 3T3 showed a time course of acidification that followed a double-exponential function with a fast and a slow component of, on average, tau=165 s and 1780 s, whereas mock 3T3 showed only a single slow tau of 1560 s. Expression of Nox1 also caused cells to acidify the extracellular medium at higher rates than control cells; Nox1 3T3 released 96+/-19 fmol h(-1)cell(-1) of acid equivalents compared to 19+/-12 fmol h(-1)cell(-1) in mock 3T3. These data show that expression of Nox1 results in a mechanism that has the capacity to rapidly acidify the cytosol and generate significant amounts of acid. No significant effect of Nox1 expression on the plasma membrane H(+) conductance was found.     

家兔膈神经呼气相电活动的观察

在52只麻醉兔和4只清醒兔上均观察到,自然呼吸时膈神经于呼气相有紧张性放电。同时引导膈肌肌电表明这种活动与膈肌紧张性的维持有关。这种电活动在切断引导电极外周段的膈神经后仍然存在。通气量不足、动脉血 PCO_2增加或 PO_2减少使其减少或消失;吸入纯氧或过度通气引起相反效应。切断窦神经或延髓腹外侧面化学敏感区局部贴敷浸有普鲁卡因的滤纸片,使膈神经呼气相放电增强;颈动脉窦区注入30—75μg NaCN 或将 pH=6的酸性人工脑脊液滤纸片贴于延髓化学敏感区,使膈神经呼气相放电减弱。本工作提示正常血气浓度及其变化经相同的外周和中枢化学感受机制同时发动两种调节过程:1.通过调节吸气肌位相性活动而调节肺通气量;2.通过调节吸气肌呼气相紧张性活动而调节肺机能余气量。     

Central vs. peripheral chemoreceptors in ventilatory stimulation by Hacetate

Intravenous infusion of Hacetate in conscious rabbits induces a greater decrease in cerebrospinal fluid (CSF) [HCO3-] and arterial CO2 partial pressure (PaCO2) than does HCl, HNO3, or Hacetate. To test whether acetate per se can stimulate central chemoreceptors, HCl- or Hacetate-acidified mock CSF was infused via the cisterna magna in conscious rabbits with catheters preimplanted under anesthesia. HCl infusion induced a greater decrease in PaCO2 refuting this hypothesis. To evaluate the role of the carotid body HCl and Hacetate were infused intravenously in an intact (CB+) and a chemodenervated group (CB-). In CB+ rabbits Hacetate infusion produced a greater decrease in PaCO2. In CB- rabbits, the fractional decrease in arterial PaCO2 was less for both acids compared with that of the CB+ rabbits, but it was significantly greater for Hacetate infusion (21.2 +/- 2.5%, mean +/- SE) than for HCl infusion (14.5 +/- 1.8%). Thus the carotid body is not necessary for the greater Hacetate ventilatory stimulation. The working hypothesis is that nonionic diffusion of Hacetate into brain or acetate replacement of HCO3- in CSF production lowers [HCO3-] near central chemoreceptors.     

Oxygen and carbon dioxide in the regulation of respiration.

When a sea-level resident ascends to a high altitude, his breathing immediately increases because of hypoxic stimulation of the peripheral chemoreceptors. In many species the aortic bodies are relatively unimportant in this response compared to the carotid bodies. When the subject stays at that altitude, his breathing increases progressively in the next few hours and days in a process termed ventilatory acclimatization and does not immediately return to control levels when hypoxia is terminated. Evidence is summarized indicating that this chronic process does not depend on the peripheral chemoreceptors or an initial respiratory alkalosis. Historical review indicates that the process of ventilatory acclimatization was initially attributed to renal excretion of plasma bicarbonate with development of a metabolic acidosis; but subsequent measurements indicated this process did not lower the arterial pH sufficiently to account for the ventilatory stimulation. More recently, ventilatory acclimatization has been attributed to accelerated removal of bicarbonate from the cerebrospinal fluid (CSF), producing a metabolic acidosis in the region of the medullary chemoreceptors; but still more recent observations indicate that this process, contrary to earlier observations, does not lower the CSF pH sufficiently to account for the ventilatory stimulation, either. Some other mechanism should be sought.     

Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway.

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.     

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia)

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.     

Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation.

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.