INTRODUCTION

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PROTEIN KINASE C (PKC), initially described in 1977 as a histone protein kinase activated by Ca²⁺, phospholipids and diacylglycerol (DAG), or phorbol esters (13), has been implicated as a common mechanism for the transduction of various extracellular signals into the cell to control many physiological processes (31). The PKC family consists of three major subgroups of isoenzymes on the basis of their molecular structure and cofactor requirements. One subgroup is composed of the classic PKC, ₁, ₂, and  isoforms, all of which share a C-2 region expressing the Ca²⁺ binding site; the other major subgroup, containing the novel PKC, , , , and µ isoforms, is devoid of the C-2 region and is active in the absence of Ca²⁺ (23). Atypical isoforms, PKC, , and , also lack the C-2 region as well as one of the repeated cys-rich zinc finger-binding motifs within the C-1 domain (24), are insensitive to phorbol ester stimulation, but can be stimulated by phosphatidylinositol-3,4,5-triphosphate (36).

PKC is very abundant in neuronal tissue, and particular isoforms such as PKC are found exclusively in the brain (23). Immunocytochemical localization studies for PKC, ₁, and ₂ isoforms in rat brain have also demonstrated heterogeneous distribution of these isoenzymes in various brain regions as well as discrete localization, suggesting putative functional implications (14, 28). A substantial body of evidence points to a critical role for PKC in both pre- and postsynaptic modulation of neuronal activity (for review, see Ref. 31). Furthermore, transient cerebral ischemia elicits excitatory amino acid release, intracellular Ca²⁺ increases, and enhanced degradation of phospholipids, all of which may lead to the formation of PKC activators such as DAG (2). Increased DAG membrane concentrations have been shown to induce PKC translocation (activation) from the soluble (cytosolic) fraction to the particulate (membrane) fraction as measured by cell fractionation (17).

The functional relevance of PKC to the control of breathing remains to be determined. Recent studies indicate that the endogenous activity of PKC modulates the excitability of expiratory bulbar neurons (11) and that PKC activation with phorbol esters increases respiratory drive potentials (6). However, it remains unclear whether PKC activity in the nucleus tractus solitarii (NTS), the first central relay for peripheral chemoreceptor afferent input, underlies components of the ventilatory response to hypoxia. We hypothesized that phorbol ester-induced PKC activation within the NTS will enhance ventilatory output, whereas PKC inhibition will attenuate hypoxic ventilatory responses in conscious, freely behaving rats.

	METHODS

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Animals. The experimental protocols were approved by the Institutional Animal Use and Care Committee. Survival experiments were performed on male Sprague-Dawley adult rats (200-350 g). In a preliminary stage, anesthesia was induced by pentobarbital sodium (Nembutal, 50 mg/kg ip), and rectal temperature was monitored by a Harvard thermal probe, with core temperature maintained at 37.5°C by a servo-controlled heating pad. A 1-cm incision of the skin was performed, and indwelling polyethylene catheters (PE-50, 0.56 mm ID, 0.88 mm OD) were surgically placed in the femoral artery and vein and advanced ~5-7 cm to reach the abdominal aorta and inferior vena cava for subsequent blood pressure measurements, arterial blood sampling, and fluid or drug administration. After the catheters were secured in the groin, they were tunneled subcutaneously, exteriorized in the dorsal aspect of the neck, flushed with a heparin-containing solution (1,000 U/ml saline), sealed with heat, and stored in a plastic cap sutured to the skin. Animals were then positioned in a stereotaxic apparatus (Kopf Instruments), and a small hole was drilled in the occipital skull. A small cannula (22 G; Plastics One, Roanoke, VA) was then surgically implanted at or in close proximity to the commissural NTS according to standard stereotaxic coordinates (13.85 mm from bregma, 0.2 mm off midline, 7.3 mm depth) (26). Adequate positioning of the cannulas was verified on completion of the experiments by administration of a pentobarbital sodium overdose to the animal, followed by 1-µl microinjection of 20% methylene blue for histological assessment (Fig. 1). After surgery, animals were allowed to recover for at least 48 h as demonstrated by return to normal feeding and sleep-waking schedules. Animals were provided with water and rat chow ad libitum, kept on a 12:12-h light-dark cycle (light onset at 0630), and at 22 ± 1°C ambient temperature for at least 1 wk of habituation before surgery and during the postsurgical recovery period. For habituation purposes, animals spent at least 1-2 h each day in a whole body plethysmographic chamber.

View larger version (164K): [in this window] [in a new window]   Fig. 1.   Representative example of a microinjection site in 1 animal. Arrow, position of cannula; darker region, diffusion area of 1 µl of methylene blue.

Ventilatory and cardiovascular recordings. Cardiorespiratory measurements were continuously acquired in the freely behaving, unrestrained animal placed in a previously calibrated 3-liter barometric chamber (Buxco Electronics, Troy, NY) by using the methods described by Bartlett and Tenney (3) and Pappenheimer (25). To minimize the effect of signal drift because of temperature and pressure changes outside the chamber, a reference chamber of equal size, in which temperature was measured by using a T-type thermocouple, was used. Environmental temperature was maintained slightly below the thermoneutral range (24-28°C). A calibration volume of 0.5 ml of air was repeatedly introduced into the chamber before and on completion of recordings. At least 60 min before the start of each protocol, animals were allowed to acclimate to the chamber, in which humidified air (90% relative humidity) was passed through at a rate of 5 l/min by using a precision flow pump-reservoir system. Pressure changes in the chamber because of the inspiratory and expiratory temperature changes were measured by using a high-gain differential pressure transducer (model MP-45-1, Validyne) (8). Analog signals were continuously digitized and were analyzed on-line by a microcomputer software program (Buxco Electronics). A rejection algorithm was included in the breath-by-breath analysis routine and allowed for accurate rejection of motion-induced artifacts. Inspiratory time, expiratory time, tidal volume, respiratory frequency, and minute ventilation (E) were computed and stored for subsequent off-line analysis.

Chemicals. Phorbol 12-myristate 13-acetate (PMA), 4-phorbol 12,13-didecanoate (4-PDD), {2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3(1H-indol-3-yl) maleimide, HCl} [bisindolylmaleimide (BIM)] I, and BIM V were all obtained from Calbiochem (La Jolla, CA).

Protocol. To determine optimal dose-responses, PMA and 4-PDD were dissolved in DMSO-normal saline (20:80) and microinjected over a period of 1 min with a microsyringe (Hamilton 7001KH, Reno, NV) at increasing concentrations ranging from 0.25 to 2 mmol/µl. For control, 1 µl DMSO-normal saline solution was microinjected before PMA or 4-PDD administration. Only one dose was employed for each animal, and cardioventilatory measurements were monitored over 4 h postinjection. For the PKC inhibitor BIM I, the concentration achieving maximal attenuation of hypoxic response during a pilot study of four rats was selected.

Measurement of blood-gas values. Arterial blood samples were obtained from the implanted arterial catheter. After withdrawal of 75-100 µl of blood in the dead space of the catheter, another 150 µl were sampled for immediate analysis of PO₂, PCO₂, and pH with a blood-gas analyzer (model 178, Ciba Corning). Measurements were always performed in room air, and during the last minute of each challenge.

Tissue lysate preparation and immunoblotting procedures. Normoxic and acutely hypoxic (15 min in 10% O₂) rats were euthanized with a pentobarbital overdose. The skull was rapidly opened, and the brain was extracted, immediately placed in ice-cold artificial cerebrospinal fluid, and dissected under surgical microscopy. The obex was visually identified, and a coronal section 1 mm caudal to 1 mm rostral to the obex was performed. The dorsal regions of the caudal brain stem were identified, carefully removed by using a 17-gauge thin-walled hypodermic needle for punch sampling, and stored at 70°C. Approximately 10 mg of tissue were obtained from each rat. For Western blot analysis, tissues corresponding to the dorsal regions of the caudal brain stem from three to five animals were pooled and homogenized at 4°C. After removal of nuclei by 5-min centrifugation at 2,000 g at 4°C, separation of the particulate and soluble fractions was performed by 1-h centrifugation at 100,000 g at 4°C.

PKC activity assay. NTS tissue homogenates obtained as described above from four normoxic or hypoxic (15 min in 10% O₂) animals were pooled and mixed in lysis buffer [(in mM) 25 Tris, pH 7.5, 2 EDTA, 0.5 EGTA, 5 1,4-dithiothreitol, and 1 phenylmethylsulfonyl fluoride, as well as leupeptin, aprotinin, and 1% Triton-X], nuclei were removed, samples were centrifuged at 100,000 g for 1 h, and both particulate and soluble fractions were partially purified through DEAE 52 cellulose anion exchange columns. PKC activity was measured with a commercially available colorimetric PKC activity assay kit (Pierce, Rockford, IL) by using a dye-conjugated glycogen synthase peptide as the substrate. PKC activity was then calculated from a concomitant standard curve assayed with purified PKC. PKC activity was corrected for protein content (DC-Bio-Rad protein assay), and results are therefore expressed as units per milligram protein.

Data analysis. Values are reported as means ± SE. Unless indicated otherwise, only experiments in which adequate cannula location in the NTS was confirmed are reported (Fig. 1). Changes in ventilatory measurements were assessed as the average of stable 3-min-period recordings for epochs before or after stimulus administration. The hypoxic response was considered as the peak E achieved over a consecutive 3-min period during the 30-min hypoxic challenge. Differences among the various treatment groups were compared by ANOVA (two-way ANOVA for repeated measures) and the Newman-Keuls test (38). Translocation of PKC subcellular fractions was determined by calculating the particulate-to-soluble fractions ratio of densitometric measurements. For each PKC isoform, ratios from bands corresponding to normoxic and hypoxic conditions were compared by unpaired t-tests. A P value of < 0.05 was considered to achieve statistical significance.

	RESULTS

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Cardioventilatory responses to PKC activation in normoxia. To determine the role of PKC in the NTS, PMA was microinjected at increasing doses, ranging from 0.25 to 2 mmol/µl. No discernible changes in behavior or in cardiovascular and ventilatory measurements occurred at 0, 0.25, and 0.5 mmol PMA (n = 6). When 1 µl of 2-mmol PMA-containing solution was injected, animals developed motor and behaviorally agitated states followed by tonic-clonic generalized seizures within ~20 min from microinjection, and they were euthanized by a lethal pentobarbital dose. PMA at 1 mmol/µl (n = 9) elicited significant cardiorespiratory enhancements within 23.6 ± 0.9 min from microinjection, and these ventilatory changes lasted for at least 4 h, at which time recordings were discontinued.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Mean (±SE) minute ventilation (E) measurements at baseline and after 1-µl nucleus tractus solitarii (NTS) microinjection of vehicle, phorbol 12-myristate 13-acetate (PMA; 1 mmol/µl), and 4-phorbol 12,13-didecanoate (4-PDD; 2 mmol/µl) as well as PMA in non-NTS sites (PMA***; 1 mmol/µl). * P < 0.01, PMA vs. vehicle (ANOVA).

View this table: [in this window] [in a new window]   Table 2.   Mean cardiorespiratory and arterial blood-gas changes after vehicle or 4-PDD microinjection in the NTS

PKC inhibition and cardiorespiratory responses to hypoxia. Hypoxic ventilatory responses were measured after 1-µl microinjection of the active PKC inhibitor BIM I (1 mmol/µl), the inactive analog BIM V (1 mmol/µl), or vehicle (Con). In Con, E increased from 139 ± 9 ml/min in room air to 285 ± 26 ml/min in 10% O₂ (Table 3, Fig. 3.). After BIM V administration, both room air and hypoxic E responses were comparable to those measured after vehicle (n = 7; Table 3, Fig. 3.; P = not significant vs. Con).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Mean (±SE) E measurements at baseline, after 1-µl NTS microinjections of vehicle ( and ), bisindolylmaleimide (BIM) I (), or BIM V (), and during hypoxic challenges (10% O2). * P < 0.02, BIM I vs. vehicle in hypoxia (ANOVA).

PKC isoform expression and activation during hypoxia in dorsal regions of the caudal brain stem of adult rats. To determine PKC isoform expression in the dorsal regions of the caudal brain stem, Western blots of whole cell lysates of NTS tissue lysates were initially performed. Immunoblots revealed the presence of all PKC isoforms tested, namely, , , , , , , , , and µ isoenzymes, in the NTS.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Western blots of soluble (S) and particulate (P) fractions of dorsal part of caudal brain stem harvested from rats during normoxia [room air (RA), left lane] and after 15 min of hypoxic challenge (HYP; 10% O2; right lane). Decreases in particulate PKC fraction and increases in particulate PKC, , , , and  fractions are apparent during hypoxia. No changes in PKCµ and  isoenzymes occurred.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Mean (±SD) PKC isoform activity changes expressed as particulate-to-soluble fractions densitometry ratios during normoxia (solid bars) and after 15 min of 10% O2 hypoxia (hatched bars). Because PKC is expressed only in particulate fraction of neurons within central nervous system (18), actual densitometry values are shown for this isoform. PKC: P < 0.002; PKC: P < 0.0001; PKC: P < 0.0005; PKC: P < 0.02; PKC: P < 0.02; PKC: P < 0.05; PKCµ, PKC, and PKC, P = not significant.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Mean (±SD) overall PKC activity in soluble and particulate fractions of dorsal part of caudal brain stem lysates harvested from animals during normoxia (solid bars) and after 15 min of hypoxic challenge (10% O2; hatched bars). Activity is expressed as U/mg protein.

	DISCUSSION

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This study demonstrates that PKC-mediated signal transduction mechanisms in the NTS substantially contribute to hypoxic ventilatory responses in the freely behaving rat. Furthermore, we provide initial evidence that suggests that both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms are activated during hypoxia within the dorsal regions of the caudal brain stem.

The NTS is the site of the first central synapse for primary afferent fibers originating from cardiopulmonary receptors, arterial baroreceptors, and chemoreceptors, as well as other visceral receptors (15). Earlier studies with retrograde tracers have revealed that the medial, dorsomedial, lateral, and commissural regions of the NTS receive the densest innervation from peripheral chemoreceptor afferent fibers. The localization of groups of neurons responsible for transmitting the afferent information through the NTS has been attempted by permanent or temporary pharmacological impairment of synaptic transmission. Housley and Sinclair (12) lesioned small areas of the NTS by microinjecting kainic acid and showed that lesions in the commissural nucleus attenuated the ventilatory response to hypoxia, whereas more rostral lesions were ineffective. Thus the NTS sites targeted in our experiments encompassed regions mediating the ventilatory response to hypoxia.

Excitatory amino acids in general, and N-methyl-D-aspartate (NMDA) glutamate receptors in particular, are primarily involved in the early hypoxic response. Reductions in E in normoxia and in the early ventilatory response to hypoxia follow NMDA-receptor blockade by intraventriculocisternal administration of the noncompetitive antagonist MK-801 in lightly anesthetized, spontaneously breathing dogs (1, 16). Similarly, glutamate microdialysate elevations occurred in the NTS of awake rats during hypoxia (20), and parenteral administration of MK-801 markedly attenuated peripheral chemoreceptor-mediated ventilatory responses to hypoxia and sodium cyanide (10).

In the NMDA glutamate receptor, intracellular loops with potential phosphorylation sites for a number of kinases including PKC have been identified (32). In addition, NMDA-receptor function may be modulated by channel phosphorylation, whereby PKC increases the probability of channel openings and also reduces voltage-dependent magnesium block of NMDA-receptor channels (7). Furthermore, activation of tyrosine kinases after intracellular elevation of free Ca²⁺ could activate PKC and has been shown to modulate NMDA-dependent whole cell currents (35). Thus, on the basis of the substantial body of evidence suggesting that PKC and NMDA glutamate-receptor activation are intimately related in several neural structures, we postulated that PKC activation would occur in relation to neural events within the NTS brought about by environmental hypoxia and consequent peripheral chemoreceptor activation.

The sequence of events leading to PKC activation appears to start with a Ca²⁺-activated translocation from the cytosol to the plasma membrane and binding to membrane phospholipids (17). Such binding induces a modification in structural configuration and allows PKC to interact with either physiological activators, such as DAG, or with pharmacological reagents, such as phorbol esters, to form a very stable active complex (22, 33). However, Ca²⁺-independent isoforms may not require Ca²⁺ binding to interact with lipid head groups because they already possess a structural conformation that permits binding of acidic lipids (22). DAG or phorbol ester binding to PKC serves as a hydrophobic anchor to the membrane, causes a dramatic increase in the enzyme's membrane affinity, stabilizes its active conformation, and results in pseudosubstrate release and maximal activation (22). The active phorbol ester PMA, but not the inactive phorbol ester 4-PDD, elicited marked increases in ventilatory output as well as significant cardiovascular effects. Moreover, these responses displayed some regional specificity, because no cardioventilatory effects emerged when PMA was administered in the cerebellum or spinal cord. Thus, although we cannot definitively rule out the possibility that PMA may have diffused to other neighboring brain stem regions and acted there to induce the long-lasting cardioventilatory effects measured in our experiments, present findings suggest that PKC activation within the NTS is associated with marked cardioventilatory enhancements in conscious rats. Our experiments also indicate that PMA has a very narrow physiological window and that more elaborate studies will be necessary to define the cellular membrane permeability and tissue diffusibility properties of PMA in vivo. Nevertheless, the effective concentration of PMA as a selective PKC activator is strikingly similar to that employed by other investigators in an in vitro preparation (30), whereas the inactive analog 4-PDD failed to elicit any measurable response even at 2 mmol/µl.

The marked attenuation of the hypoxic ventilatory response in rats treated with BIM I, the translocation of particular PKC isoforms, and the overall increase in PKC activity in the particulate fraction of NTS lysates in hypoxia further indicate that both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms are activated in the NTS during hypoxia and that this increase in PKC activity underlies important components of the hypoxic ventilatory response. However, determination of the role played by each of the PKC isoforms and the various time frames of such PKC isoform activation clearly await further studies. It is important to note that the application of BIM I or BIM V to the NTS did not modify any of the ventilatory measurements in our conscious rats during normoxia. We interpret these findings as indicative that PKC does not play a role in maintaining neuronal excitability within the NTS. Alternatively, if such a role is indeed present, compensatory mechanisms may be activated and may counterbalance the effect elicited by application of the PKC inhibitor.

The concentrations of BIM I employed in this study were selected on the basis of preliminary studies and corresponded to the lowest dose above which no further inhibitory effect on hypoxic ventilatory response occurred. However, such an approach raises the possibility that the activity of other kinases, such as protein kinase A or tyrosine kinases, may have been affected (34). This is an unlikely possibility because BIM I is highly selective for PKC and BIM I half-maximal inhibitory concentration for PKC is 200-fold lower than for PKA or receptor tyrosine kinases. In addition, one has to consider that the final concentrations of the drugs in the NTS tissue were decreased by the efficiency of diffusion through the tissue by at least one to two orders of magnitude (4). Thus we believe that the BIM I dose used in this study may have a selective action on PKC.

Membrane currents of cardiovascular neurons within the rostral ventrolateral medulla were not modified by PKC inhibition in response to hypoxia (29). In contrast, neuronal responses were abolished when a baroreceptor stimulus was applied (29). Thus signal transduction pathways of rapidly developing excitatory currents within hypoxic chemosensititive rostral ventrolateral medulla neurons are not mediated by PKC. In cats, PKC modulates basal activity and excitability of expiratory neurons within the ventral respiratory group (11). Because the excitatory synaptic drive of these expiratory neurons is primarily dependent on glutamate via NMDA and DL--amino-3-hydroxy-5-methylisoxazole-propionic acid/quisqualate receptors (27), excitatory cationic inward currents are under PKC-mediated tonic modulatory control (11). Present experiments further indicate that PKC activation in the NTS of freely behaving rats mediates tonic and chemosensitive excitatory respiratory drives during normoxia and hypoxia. It remains unclear, however, whether different PKC isoforms are responsible for modulation of intrinsic and synaptic excitability of NTS neurons.

Transient activation of Ca²⁺-dependent PKC occurs during neural tissue ischemia and is then followed by decreases in PKC activity (5, 37). Similarly, brain ischemia by transient middle cerebral artery occlusion was shown to elicit selective PKC induction, which was inhibited by NMDA glutamate-receptor blockade (19). In contrast, in a nonneural system such as cardiac myocytes, isoform-selective PKC subcellular redistribution occurred in response to O₂ deprivation (9). In this latter experimental setting, 1 h of severe hypoxia induced increases in the particulate fraction of PKC and PKC, whereas PKC immunoreactivity increased in the soluble fraction (9). Thus, from such limited data, it would appear that the pattern of subcellular fraction PKC redistribution with O₂ limitation will vary from tissue to tissue. The decrease in the particulate fraction of PKC at 15 min of a mild to moderate hypoxic challenge within the dorsal part of the caudal brain stem was not anticipated, because overall PKC activity increased during hypoxia. PKC and PKC, the other Ca²⁺-dependent PKC isoforms, displayed increases in the particulate fraction, a feature typically considered as indicative of PKC activation. Thus we had expected that PKC would also be recruited to the membrane fraction. It is possible that PKC activation occurs at an earlier stage in the hypoxic challenge and is related to the process of NMDA-receptor channel opening. Alternatively, PKC could be playing a role in the maintenance of NMDA channel closure, such that reduction in PKC membrane activity would facilitate channel opening. Translocation of PKC to the nucleus could have occurred and would have therefore been missed by our subcellular fractionation strategy. Finally, it has recently become apparent that protein-protein interactions of PKC with anchoring proteins may alter the phospholipid-dependent translocation of PKC isoforms and/or lead to preferential intracellular targeting (21). Of the remaining PKC isoforms displaying translocation to the membrane in the dorsal portion of the caudal brain stem during brief hypoxia, members of all three PKC family subclasses, classic (PKC and PKC), novel (PKC and PKC), and atypical (PKC), were represented and showed increased abundance in the particulate fraction during hypoxia. These findings are further supported by the overall increase in PKC activity measured in the particulate fraction of the dorsal portion of the caudal brain stem of hypoxic rats (Fig. 6). In contrast, cortical regions displayed a different pattern of PKC activation with hypoxia (data not shown). Thus it is conceivable that PKC translocations in the dorsal portion of the caudal brain stem during hypoxia may underly signal transduction events related to synaptic activity changes associated with enhanced afferent inputs from stimulated peripheral chemoreceptors and may also represent direct, nonspecific effects of hypoxic stress on neuronal populations contained within the dorsal portion of the caudal brain stem or other brain structures. It should be emphasized at this point that tissue lysates obviously contain multiple cell types, such as neurons, glia, fibroblasts, and other nonneuronal cell types, that could be contaminating our data, such that no firm conclusions regarding PKC isoform translocation patterns within a particular cell type can be drawn at this point. Delineation of PKC isoforms involved in the second-messenger pathways of a specific cell type and resulting from the involvement of the two different components of the hypoxic stimulus, i.e., stress and synaptic transmission changes, will require further study.

In summary, PKC activation within the NTS induces ventilatory increases during normoxia, whereas PKC inhibition attenuates hypoxic ventilatory responses in unrestrained conscious rats. Increases in overall PKC activity were measured during hypoxia in the particulate fraction. Although all PKC isoforms tested were expressed in the NTS, only PKC, , , , and  isozymes showed translocation to the particulate fraction, whereas PKC immunoexpression was increased in the soluble fraction. We conclude that, within the NTS, PKC underlies critical components of both tonic and hypoxic chemotransducive ventilatory drives.