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J Appl Physiol 84: 641-648, 1998;
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Cardiorespiratory responses to systemic administration of a protein kinase C inhibitor in conscious rats

David Gozal, Gavin R. Graff, José E. Torres, Sanjay G. Khicha, Gautam S. Nayak, Narong Simakajornboon and Evelyne Gozal

Constance S. Kaufman Pediatric Pulmonary Research Laboratory, Departments of Pediatrics and Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Gozal, David, Gavin R. Graff, José E. Torres, Sanjay G. Khicha, Gautam S. Nayak, Narong Simakajornboon, and Evelyne Gozal. Cardiorespiratory responses to systemic administration of a protein kinase C inhibitor in conscious rats. J. Appl. Physiol. 84(2): 641-648, 1998.---Although protein kinase C (PKC) is an essential component of multiple neurally mediated events, its role in respiratory control remains undefined. The ventilatory effects of a systemically active PKC inhibitor (Ro-32-0432; 100 mg/kg ip) were assessed by whole body plethysmography during normoxia, hypoxia (10% O2), and hyperoxia (100% O2) in unrestrained Sprague-Dawley rats. A sustained expiratory time increase occurred within 8-10 min of injection in room air [mean 44.8 ± 5.2 (SE) % ], was similar to expiratory time prolongations after Ro-32-0432 administration during 100% O2 (45.5 ± 8.1%; not significant), and was associated with mild minute ventilation (VE) decreases. Hypercapnic ventilatory responses (5% CO2) remained unchanged after Ro-32-0432. During 10% O2, VE increased from 122.6 ± 15.6 to 195.7 ± 10.1 ml/min in vehicle-treated rats (P < 0.001). In contrast, marked attenuation of VE hypoxic responses occurred after Ro-32-0432 [86.2 ± 6.2 ml/min in room air to 104.1 ± 7.1 ml/min in 10% O2; pre- vs. post-Ro32-0432, P < 0.001 (analysis of variance)]. Overall, PKC activity was reduced and increases with hypoxia were abolished in the particulate subcellular fraction of brain tissue after Ro-32-0432 treatment, indicating that this compound readily crosses the blood-brain barrier. We conclude that systemic PKC inhibition elicits significant centrally mediated expiratory prolongations and ventilatory reductions as well as blunted ventilatory responses to hypoxia but not to hypercapnia. We postulate that PKC plays an important role in signal transduction pathways within brain regions underlying respiratory control.

hypoxia; hypercapnia; respiratory control; signal transduction; N-methyl-D-aspartate receptor

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PROTEIN KINASE C (PKC) is an ubiquitous histone protein kinase activated by Ca2+, phospholipids and diacylglycerol (DAG), or phorbol esters (16). Since PKC identification by Inoue and colleagues (16), this family of enzymes has been implicated as an important signal transduction pathway for many cellular and physiological events (22, 27). PKC is very abundant in neuronal tissue, and particular isoforms such as PKCgamma are exclusively found in the brain (22). The heterogeneous topographical distribution of PKC isoforms within particular brain regions is suggestive of functional specificity (17, 24); for example, genetically engineered mice lacking PKCgamma demonstrate marked deficits in memory and learning abilities (1).

The ventilatory response to hypoxia in rats is critically dependent on excitatory amino acids in general (9, 25) and more specifically on activation of N-methyl-D-aspartate (NMDA) receptors (13, 15, 20). The intracellular domain of NMDA receptors contains multiple consensus sequences for serine/threonine phosphorylation that could activate kinases such as PKC or Ca2+-calmodulin-dependent kinase type II (21). Furthermore, PKC has been shown to modulate NMDA-receptor channel opening (28, 29). Thus PKC-mediated pathways could underlie important components of the hypoxic ventilatory response via its modulatory effects on NMDA-receptor function.

The functional relevance of PKC to respiratory control remains unclear. Present available evidence suggests that endogenous PKC activity modulates the excitability of expiratory bulbar neurons (14) and that phorbol ester-induced PKC activation is associated with increases in respiratory drive potentials (7). In addition, in vivo PKC depletion by prolonged application of phorbol esters to superficial regions within the ventrolateral medulla appears to attenuate the ventilatory response to inhaled CO2 (11). The recent development of a highly selective PKC inhibitor that may be administered systemically (5) prompted us to examine whether PKC inhibition modifies normoxic ventilation or alters hypoxic and hypercapnic ventilatory responses in conscious freely behaving rats.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

The experimental protocols were approved by the Institutional Animal Use and Care Committee. Survival experiments were conducted on male Sprague-Dawley adult rats (200-350 g). In a first 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 at the femoral crease was performed, and indwelling polyethylene catheters (PE-50, 0.56 mm ID, 0.88 mm OD) were surgically introduced 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 allowed to recover for at least 48 h after surgery, 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 plethysmograph chamber.

Ventilatory and Cardiovascular Recordings

Cardiorespiratory measures were continuously acquired in the freely behaving, unrestrained rat placed in a previously calibrated 3-liter barometric chamber (Buxco Electronics, Troy, NY) by using the methods described by Bartlett and Tenney (4) and Pappenheimer (23). To minimize the effect of signal drift due to 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 within the thermoneutral range (24-28°C). A calibration volume of 1.0 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 8 l/min by using a precision flow pump-reservoir system. Pressure changes in the chamber due to the inspiratory and expiratory temperature changes (12) were measured by using a high-gain differential pressure transducer (model MP45-1, Validyne). Analog signals were continuously digitized and analyzed online 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. One-minute epochs associated with >30% rejection were not considered in the analysis. Inspiratory time (TI), expiratory time (TE), tidal volume (VT), respiratory frequency, and minute ventilation (VE) were computed and stored for subsequent off-line analysis.

Systemic arterial blood pressure was measured from the arterial femoral catheter connected to a calibrated pressure transducer via a custom-designed swivel apparatus in the recording chamber (Buxco Electronics). Physiological signals were digitized, and a beat-to-beat peak-trough analysis routine allowed computation of heart rate (HR), systolic (SBP) and diastolic (DBP) blood pressure, and mean arterial pressure (MAP) values.

Protocol

Stage 1. Ro-32-0432 is a bisindolylmaleimide derivative, in which, besides a straight-chain alkyl side chain bearing a cationic substituent, the position of the amine substituent was correctly conformationally restricted (6). To determine the optimal dosage for our experiments, Ro-32-0432 (Roche Products, Welwyn Garden City, UK) was dissolved in normal saline and injected intraperitoneally at increasing dosages, ranging from 0 to 200 mg/kg. Only one dose was employed for each animal, and cardioventilatory measurements were monitored over 4 h postinjection. The lowest dose achieving maximal prolongation of expiratory duration during this pilot study in four animals in each group (100 mg/kg) was selected for subsequent experiments.

Stage 2. After the optimal PKC-antagonist dosage was determined, cardiac and respiratory responses to either vehicle or Ro-32-0432 were assessed in 16 rats breathing room air.

Stage 3. Cardioventilatory responses to hypoxia in 16 rats (10% O2-balance N2 for 30 min) were determined 30 min after vehicle or Ro-32-0432 administration. At least 2 h of recovery in normoxia were allowed between hypoxic challenges.

Stage 4. To elucidate whether the differences in normoxic and hypoxic ventilation were due to a peripheral or central effect by Ro-32-0432, physiological responses to Ro-32-0432 and vehicle were measured in 16 additional rats during hyperoxia (100% O2) or normoxia.

Stage 5. To determine whether PKC inhibition modifies the ventilatory response to hypercapnia, six rats underwent ventilatory challenges with 5% CO2-balance room air before and after Ro-32-0432 administration.

Tissue Lysate Preparation and PKC Activity Assay

To ascertain that Ro-32-0432 crosses the blood-brain barrier, normoxic and acutely hypoxic (30 min in 10% O2) rats were euthanized with a pentobarbital sodium 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 frontoparietal cortex was visually identified, removed, and stored at -70°C. Tissue homogenates were prepared in lysis buffer {[(in mM) 25 tris(hydroxymethyl)aminomethane, pH 7.5, 2 EDTA, 0.5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5 1,4-dithiothreitol, and 1 phenylmethylsulfonyl fluoride], as well as leupeptin, aprotinin, and 1% Triton X-100} and 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 use of 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, and results are therefore expressed as units per milligram protein.

Data Analysis

Values are reported as means ± SE unless indicated otherwise. Changes in ventilatory measurements were assessed as the average of stable 3-min-period recordings for epochs preceding stimulus administration and 3-min periods corresponding to peak responses. Differences among the various treatment groups were compared by analysis of variance (ANOVA; 2-way ANOVA for repeated measures) and the Newman-Keuls test (31). A P value of < 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Ventilatory Responses

Dose-response curves to Ro-32-0432 on ventilatory measurements are shown in Fig. 1. Significant prolongations of TE occurred at 25 mg/kg and peaked at 100 mg/kg, with no further increases thereafter. Thus the dose of 100 mg/kg was retained for all subsequent experiments.


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  		Fig. 1.   Ventilatory changes (Delta ) induced by administration of increasing doses of protein kinase C (PKC) inhibitor Ro-32-0432 (0-200 mg/kg ip). Each data point represents mean ± SE of 4 different animals. TI, inspiratory time; TE, expiratory time; VT, tidal volume; f, breathing frequency; VE, minute ventilation.

Ro-32-0432 was associated with significant TE prolongations (Fig. 2; 0.369 ± 016 to 0.519 ± 0.016 s; P < 0.001), whereas vehicle injection did not modify TE (0.345 ± 0.021 to 0.340 ± 0.017 s; P = not significant). VT was not changed after Ro-32-0432 administration, such that VE decreased by 28.9 ± 3.1% (P < 0.01) and was associated with mild, albeit significant, increases in arterial PCO2 (PaCO2) (Table 1).


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  		Fig. 2.   Ventilatory responses to hypoxia (10% O2) at 10 and 30 min in 16 vehicle- and Ro-32-0432-treated rats (100 mg/kg ip). Values are means ± SE. B1, baseline 1; V, vehicle; B2, baseline 2; Tx, Ro-32-0432; black-square, TI; open circle , TE.

                              
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  		Table 1.   Mean cardiorespiratory and arterial blood gas changes in 16 rats after Ro-32-0432 injection during normoxia and hypoxia

When Ro-32-0432 was administered to 16 additional rats randomly assigned to either room air or hyperoxia, similar changes in ventilatory measurements occurred during both inspired O2 fraction conditions (Fig. 3, Table 2). These findings suggest that Ro-32-0432 had minimal if any effect on peripheral chemoreceptor afferent input.


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  		Fig. 3.   Ventilatory changes induced by Ro-32-0432 (100 mg/kg ip). Values are means ± SE; n = 8 rats during normoxia [room air (RA)] and 8 rats exposed to hyperoxia (100% O2). open circle , TI; black-triangle, TE.

                              
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  		Table 2.   Mean cardiorespiratory and arterial blood-gas changes after Ro-32-0432 administration during normoxia and hyperoxia

Vehicle-treated animals mounted brisk ventilatory responses to hypoxia, with VE increases of 60.2 ± 5.5% (Fig. 2, Table 1). In contrast, after Ro-32-0432 treatment, significant attenuation of the VE response occurred [22.2 ± 5.0%; P < 0.001 (2-way ANOVA)].

In contradistinction to the hypoxic ventilatory responses, no attenuation of hypercapnic responses occurred after Ro-32-0432 administration (Fig. 4, Table 3).


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  		Fig. 4.   Ventilatory responses to hypercapnia (5% CO2). Values are means ± SE; n = 6 vehicle (black-square)- and Ro-32-0432-treated rats (100 mg/kg ip; open circle ).

                              
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  		Table 3.   Mean cardiorespiratory and arterial blood gas changes after Ro-32-0432 administration during room air and 5% CO2

Cardiovascular Responses

During normoxia, Ro-32-0432 elicited significant HR and MAP decreases compared with vehicle (Fig. 5, Table 1). However, the chronotropic response to hypoxia was preserved after the administration of the PKC inhibitor, such that HR increased by 8.9 ± 1.1 and 6.9 ± 1.0% in vehicle- and Ro-32-0432-treated animals, respectively. Similarly, the mild MAP decrease traditionally observed during hypoxia (-12.6 ± 2.1%) was also present after Ro-32-0432 (-9.4 ± 1.6%; P = not significant; Fig. 5). Cardiovascular responses during hypercapnia were also unaffected by PKC inhibition (Table 3).


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  		Fig. 5.   Heart rate (HR) and mean arterial pressure (MAP) responses to hypoxia (10% O2; n = 16) and hypercapnia (5% CO2; n = 6) in vehicle (black-square)- and Ro-32-0432-treated rats (100 mg/kg ip; open circle ). Values are means ± SE.

Overall Brain PKC Activity

Ro-32-0432 was associated with marked decreases of PKC activity in the soluble and particulate subcellular fractions of neocortical tissue lysates (Fig. 6). In addition, the increase in PKC activity of the particulate fraction induced by environmental hypoxia was prevented by Ro-32-0432 (Fig. 6).


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  		Fig. 6.   Overall PKC activity changes in particulate (solid bars) and soluble (open bars) fractions of neocortical brain tissue lysates induced by administration of Ro-32-0432 during normoxia and hypoxic challenges. Values are means ± SE.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Our study shows that systemic administration of Ro-32-0432, a potent and highly selective inhibitor of PKC, is associated with significant TE prolongations and ventilatory reductions during normoxia as well as with mild to moderate decreases in MAP and HR in freely behaving rats. Furthermore, although Ro-32-0432 failed to modify hypercapnic ventilatory responses, it markedly attenuated the ventilatory response to hypoxia.

Activation of PKC is primarily regulated by the formation of DAG via phospholipid hydrolysis and by changes in intracellular Ca2+, which may originate from both extracellular and intracellular sources. In the mammalian central nervous system, available evidence suggests that PKC plays a prominent role in the processing of neuronal signals and in the short- or long-term modulation of synaptic transmission (27). This kinase is a member of a family consisting of at least 11 described isoenzymes. The homologous structure of each isoform makes resolution of the enzymological properties of the enzyme difficult because more than one isoform may be expressed in a particular neuron. Thus the distinct functional roles of PKC subspecies in mammalian tissues have so far been inferred from the tissue localization of each isoform. Ro-32-0432 is a systemically administrable highly selective inhibitor of PKC with >= 100-fold selectivity for PKC compared with a variety of serine/threonine- and tyrosine-specific protein kinases (5). Similar to other bisindolylmaleimide derivatives, Ro-32-0432 competes with ATP for binding to the catalytic domain of PKC (10). In addition, Ro-32-0432 displays higher selectivity for Ca2+-dependent PKC isoforms such as PKCalpha , beta I, beta II, and gamma , compared with novel Ca2+-independent isoenzymes such as PKCepsilon (5, 30). However, the degree of isoform selectivity by Ro-32-0432 does not allow for any precise conclusions to be drawn about the role of each PKC isoform in our present study.

An important methodological issue in this study concerned the permeability of the blood-brain barrier to Ro-32-0432. Formation of increasing DAG membrane concentrations has been shown to induce PKC translocation (activation) from the soluble (cytosolic) fraction to the particulate fraction (membrane) as measured by cell fractionation (19). Thus increases in PKC activity would be more readily identifiable in particulate subcellular fractions rather than in whole-cell lysates. Overall PKC activity was reduced in both soluble and particulate fractions of neocortical tissue, and the increase in PKC activity with hypoxia in the particulate subcellular fraction was prevented by Ro-32-0432, thereby indicating that this compound readily crosses the blood-brain barrier.

Ventilatory Responses

Ro-32-0432 elicited dose-dependent TE prolongations, which resulted in significant respiratory frequency and VE reductions. In contrast, VT was not affected. Our results are in close agreement with those of Haji et al. (14), who found that PKC inhibition reduced neuronal excitability of expiratory neurons in the ventral respiratory group in cats. Because PKC is ubiquitously distributed, inhibition of this enzyme by Ro-32-0432 could have impacted on the metabolic rate in the animals, leading to VE reductions commensurate with the magnitude of the metabolic decrease. However, PaCO2 was increased after Ro-32-0432 administration, suggesting that ventilatory drive was affected by this compound, irrespective of the metabolic consequences of systemic PKC blockade.

The similarity of ventilatory responses to Ro-32-0432 in normoxic and hyperoxic rats further suggests that central PKC inhibition was primarily responsible for such ventilatory changes and that PKC inhibition of peripheral structures and/or afferent inputs was not a major contributor to such responses. However, the effect of PKC inhibition on contributions from other visceral or chest wall afferent inputs cannot be ruled out at this time. In addition, carotid sinus neurograms were not specifically measured, and we were obviously limited by virtue of our experimental design in assessing which brain structures were primarily affected by PKC inhibition. Nevertheless, our results suggest that endogenous PKC activity in conscious rats plays a preponderant role in mechanisms associated with respiratory rhythm generation and that neuronal populations underlying volume control do not exhibit PKC dependency.

The ventilatory response to elevations in inspired CO2 remained unchanged after administration of the PKC inhibitor. In contrast, Dreshaj and colleagues (11) have recently reported preliminary evidence in anesthetized piglets suggesting that PKC activity may underlie important functional excitatory components of neuronal activation within ventrolateral medullary neurons that exhibit CO2-chemosensitive properties. However, these investigators allowed for long-term (~4 h) phorbol ester applications to the medullary neurons to achieve PKC downregulation. Such strategy could have induced significant cellular damage to these neurons, and, therefore, the diminished CO2 responses that followed such phorbol ester treatment could reflect cellular viability issues rather than intrinsic changes in chemosensitivity. We should point out that although the slope of the hypercapnic response was not modified by PKC inhibition, a mild, albeit significant, elevation in PaCO2 occurred with Ro-32-0432 administration, indicative of a shift to the right of the VE-PaCO2 relationship that could be construed as some degree of attenuation in chemosensitivity. Further studies are clearly needed to elucidate the role of PKC in central CO2-chemosensitive pathways.

In contradistinction to hypercapnic ventilatory responses, PKC inhibition resulted in marked attenuation of hypoxic ventilatory responses. It is now clear that excitatory amino acids, and in particular NMDA glutamate receptors, are primarily involved in the early hypoxic response. Intraventriculocisternal administration of the noncompetitive NMDA-receptor channel antagonist MK-801 reduced VE in normoxia as well as the early ventilatory response to hypoxia in lightly anesthetized spontaneously breathing dogs (2, 18). Similarly, glutamate microdialysate elevations occur within the nucleus of the solitary tract in awake rats during hypoxia (20), and parenteral administration of MK-801 markedly attenuated peripheral chemoreceptor-mediated ventilatory responses to hypoxia and sodium cyanide in conscious rats (13). Furthermore, 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 (8). In addition, transient neuronal events that elicit excitatory amino acid release are associated with intracellular Ca2+ increases and enhanced degradation of phospholipids, all of which may lead to the formation of PKC activators such as DAG (3). 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 elicited by environmental hypoxia. As a corollary of such a hypothesis, PKC inhibition would result in attenuation of hypoxic responses, which indeed occurred. Because of the obvious limitations inherent in in vivo models and systemic drug administration, we are precluded from drawing any firm conclusions regarding structural-functional relationships. Nonetheless, although the present study does not elucidate which neuronal populations underlie PKC inhibition-mediated effects, it may indicate that PKC plays an important role in respiratory control, primarily linked to central hypoxic defense mechanisms.

Cardiovascular Responses

Significant HR and MAP reductions occurred after administration of Ro-32-0432. However, the magnitude and pattern of cardiovascular responses to hypoxia and hypercapnia were preserved after PKC inhibition, thereby suggesting that endogenous PKC activity primarily modulates tonic drive to neural regions underlying both cardiac and pressor functions. In contrast, PKC does not appear to mediate changes in activity within neuronal populations underlying adaptive cardiovascular regulatory processes in response to ventilatory challenges.

Our findings concur with those reported by Sun and Reis (26), who found that membrane currents of cardiovascular neurons within the rostral ventrolateral medulla were not modified by PKC inhibition in response to hypoxia. In contrast, neuronal responses were abolished when a baroreceptor stimulus was applied (26). Thus signal transduction pathways of rapidly developing excitatory currents within hypoxia-chemosensitive rostral ventrolateral medullary neurons appear not to be mediated by PKC.

In summary, endogenous PKC inhibition is primarily associated with reduced tonic respiratory rhythm and cardiopressor drives and with marked attenuations in hypoxic ventilatory response, with no discernible effect on hypercapnic or cardiovascular responsivity. Further studies employing PKC isoform-selective compounds and targeting of functionally distinct neural structures will be necessary to define the role of PKC in cardiorespiratory control.

    ACKNOWLEDGEMENTS

We gratefully thank Roche Products for the generous gift of Ro-32-0432.

    FOOTNOTES

This study was supported in part by National Institute of Child Health and Human Development Grant HD-01072, Maternal and Child Health Bureau Grant MCJ-229163, American Lung Association Grant CI-002-N, and a postdoctoral fellow research grant from the Louisiana Chapter of the American Lung Association/American Thoracic Society.

Address for reprint requests: D. Gozal, Section of Pediatric Pulmonology, Dept. of Pediatrics, SL-37, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: dgozal{at}tmcpop.tmc.tulane.edu).

Received 31 May 1997; accepted in final form 2 October 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References


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The Journal of Applied Physiology 84(2):641-648
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