INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CYCLOOXYGENASE (COX) is the rate-limiting enzyme in the biosynthesis of prostanoids (PGs and thromboxanes). COX-derived metabolites, such as prostanoids and superoxide anions, play an important role in regulating cerebral blood flow in the newborn. For instance, prostanoids contribute to maintaining cerebral blood in hypotensive piglets (17, 18). In addition, prostanoids have a permissive effect on hypercapnia-induced pial arteriolar vasodilation (19, 20).

COX also plays an important role in the pathophysiology of cerebral circulation after ischemic stress. In the piglet cerebral cortex, COX is the source of most of the superoxide radicals detected in the early reperfusion period after total cerebral ischemia (1). Cerebral ischemia-reperfusion results in severe alterations in cerebrovascular dilator responses. For instance, hypoxic/ischemic stress transiently attenuates N-methyl-D-aspartate (NMDA)-induced vascular dilation (3-5). This response is neuronally mediated and may play a role in coupling cerebral blood flow to brain metabolism. More importantly, NMDA-induced vasodilation can be used as a sensitive bioassay to assess the functional integrity of the neuronal-vascular axis. After cerebral ischemia, NMDA-induced vasodilation has been shown to be preserved by pretreatment with oxygen free radical scavengers, inhibitors of COX, and most recently, inhibitors of protein synthesis (3-5, 28). However, the mechanism by which inhibitors of protein synthesis [both actinomycin D and cycloheximide (CHX)] preserved the vascular response to NMDA after ischemia has not been elucidated.

In this study we tested the hypothesis that CHX inhibits COX activity in the cerebral cortex and vasculature of newborn pigs. We used the COX-dependent vascular responses to arterial hypercapnia and arterial hypotension as in vivo bioassays to assess the effect of CHX on active COX levels. In addition, we investigated the effect of CHX in the conversion of exogenous arachidonic acid (AA) to PGF₂ and prostacyclin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. In these experiments newborn piglets of either sex (1- to 7-days old, 1-2 kg body wt, n = 31) were used. All procedures were approved by the Institution Animal Care and Use Committee. The animals were anesthetized with thiopental sodium (30-40 mg/kg ip) followed by intravenous injection of -chloralose (75 mg/kg). Supplemental doses of -chloralose were given to maintain a stable level of anesthesia. The right femoral artery and vein were catheterized to record blood pressure and to administer drugs and fluids, respectively. The piglets were intubated via tracheotomy and artificially ventilated with room air. The ventilation rate (~20 breaths/min) and tidal volume (~20 ml) were adjusted to maintain arterial blood gas values and pH in the physiological range; for instance, in the 1 mg/kg CHX-treated group (n = 9) the values were (means ± SE) the following: arterial pH (7.50 ± 0.02), PCO₂ (34 ± 2 mmHg), and PO₂ (96 ± 5 mmHg).

Measurement of pial arteriolar responses. Instrumented piglets (n = 22) were divided into three groups: 1) vehicle-treated control, 2) animals treated with 0.3 mg/kg CHX, and 3) animals treated with 1 mg/kg CHX. We examined the responses of cerebral arterioles to arterial hypercapnia, arterial hypotension, and sodium nitroprusside (SNP) before and 20 min after drug treatment. Hypercapnia was elicited by artificially ventilating the animal with a gas mixture containing (5% or 10% CO₂, 21% O₂, balance N₂). Arterial hypotension was induced by withdrawing venous blood to yield ~25 and 40% decreases in mean arterial pressure (MAP), respectively. After the measurements the heparinized blood was reinfused. SNP (10⁶ and 10⁵ mol/l) dissolved in aCSF was administered topically through the injection ports of the cranial window onto the brain surface with a single application. Arteriolar diameters were measured continuously for 5 min for each stimulus until steady-state values were obtained. Typically, we obtained data for two different stimuli in each animal. With this protocol we obtained similar arteriolar responses to hypercapnia, hypotension, and SNP as in previous experiences with these challenges. These doses of hypercapnia, hypotension, and SNP were chosen to provide medium and large increases in vascular diameters. Between different stimuli the window was flushed with aCSF, and the arteriolar diameters returned to baseline values. CHX (0.3-1 mg/kg) was dissolved in 3 ml of saline and injected intravenously. Twenty minutes after treatment, challenges of hypercapnia, arterial hypotension, and SNP were repeated according to the procedure described above.

Assessment of cortical PG-synthesizing capacity. We determined cortical conversion of exogenous AA to PGF₂ and 6-keto-PGF₁ (n = 9). AA (1, 10, and 20 µg/ml) dissolved in aCSF was administered onto the brain surface through the injectable ports of the cranial window. Each dose of AA was applied to the brain surface for 10 min and then the cranial window was gently flushed and the effluent aCSF (~300 µl) was collected and frozen. AA was applied at 1-h intervals twice before and then 20 min and 1 h after CHX (1 mg/kg iv) treatment. Typically, we applied AA three times in each animal. Because the data obtained from these animals did not differ significantly, we combined these data as shown in RESULTS. From the aCSF samples we determined concentrations of PGF₂ and 6-keto-PGF₁ using ELISA kits (Oxford Biomedical Research, Oxford, MI).

Statistics. Data are expressed as means ± SE. Data were analyzed using repeated measures ANOVA, and one-way ANOVA was used for differences between treatment groups. Pairwise comparisons were made using the Student-Newman-Keuls test where appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Arterial blood pressure was within normal limits (Table 1) and did not change significantly during hypercapnia. CHX administration caused a transient increase (5-15 mmHg) in MAP, but blood pressure values returned to baseline within 10 min of CHX administration.

Graded hypercapnia induced by ventilating the animals with gas mixtures containing either 5 or 10% CO₂ resulted in a concentration-dependent increase in pial arteriolar diameters in accordance with the elevated PCO₂ levels in all groups of animals (Table 1). In the vehicle-treated control group, repeated exposure to high PCO₂ levels elicited essentially identical vasodilation in pial arterioles. However, in the groups intravenously injected with either 0.3 or 1.0 mg/kg CHX, vasodilation was significantly reduced to either level of hypercapnia (Table 1, Fig. 1). The attenuation of the response was larger in the group treated with the higher dose of CHX, especially at the lower level of hypercapnia (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of cycloheximide (CHX) treatment on cyclooxygenase (COX)-dependent vascular responses. Data are plotted as the percent preservation of vascular dilation to arterial hypercapnia and hypotension after CHX treatment. In the vehicle-treated control group pial arteriolar responses to inhalation of 5-10% CO2 (5% CO2 and 10% CO2, respectively) or graded hypotension [hypotension (grade 1 and grade 2, respectively)] were fully preserved. Treatment with 0.3-1 mg/kg CHX significantly reduced vasodilation to both doses of hypercapnia. Similarly the vascular response to arterial hypotension was also reduced by CHX, and the higher dose of CHX attenuated the response >70%. * Significantly different from control group (P < 0.05).

Graded arterial hypotension induced by venous blood withdrawal resulted in a dose-dependent increase in pial arteriolar diameters in accordance with decreased arterial blood pressure levels (Table 1). In the vehicle-treated control group pial arteriolar responses in response to stimulation by arterial hypotension were unaltered (Fig. 1). The vasodilatory response to arterial hypotension was also largely retained in the group treated with 0.3 mg/kg CHX. However, arteriolar responsiveness to arterial hypotension was severely reduced in the animals treated with 1 mg/kg CHX (Table 1, Fig. 1).

SNP (n = 7) induced dose-dependent pial arteriolar vasodilation that was unaffected by treatment with 1 mg/kg CHX. Baseline arteriolar diameters were not significantly different before and after CHX treatment (112 ± 6 vs. 118 ± 7 µm, respectively), and the percent changes were 21 ± 4 vs. 19 ± 4% at 10⁶ mol/l and 38 ± 5 vs. 37 ± 5% at 10⁵ mol/l.

Topical application of 1, 10, and 20 µg/ml AA onto the brain surface elicited a dose-dependent increase in the aCSF concentrations of PGF₂ and 6-keto-PGF₁ (Figs. 2 and 3). Repeated application of AA resulted in similar changes in aCSF PG levels. CHX (1 mg/kg iv) attenuated baseline as well as AA-stimulated PGF₂ levels as early as 20 min after CHX administration (Fig. 2). The inhibition of PGF₂ synthesis lasted at least as long as 1 h after CHX administration. Similarly, AA-stimulated 6-keto-PGF₁ levels were also significantly reduced 20 min after administration of CHX (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of CHX treatment on conversion of exogenous arachidonic acid (AA) to PGF2. Topical application of 1-20 µg/ml AA resulted in concentration-dependent, reproducible increases in PGF2 concentrations in the aCSF (control 1 and control 2). In contrast, 20 min after CHX (1 mg/kg iv) treatment, baseline and AA-stimulated PGF2 levels were significantly decreased compared with control values. At 1 h after CHX treatment we obtained similar results, and there was a trend toward further inhibition of PGF2 synthesis. * Significantly different from respective control values (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of CHX treatment on conversion of exogenous AA to 6-keto-PGF1. Topical application of 1-20 µg/ml AA resulted in concentration-dependent, reproducible increases in aCSF concentrations of 6-keto-PGF1 (control 1 and control 2). In contrast, 20 min after CHX (1 mg/kg iv) treatment, AA-stimulated 6-keto-PGF1 levels were significantly decreased compared with control values. At 1 h after CHX treatment we obtained similar results, and there was a trend toward further inhibition of 6-keto-PGF1 synthesis. * Significantly different from respective control values (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major new finding in this study is that in vivo CHX rapidly inhibits COX activity in the piglet cerebral cortex and vasculature. More specifically, cerebrovascular reactivity to COX-dependent, vasodilatory stimuli is diminished within 20 min of CHX administration. Similarly, cerebral cortical PG-synthesizing capacity is largely reduced shortly after CHX treatment.

The most likely mechanism of how CHX inhibits COX activity in our experimental model is through its inhibitory effect on protein translation. Although we did not determine the effect of 0.3-1 mg/kg CHX on cortical protein synthesis, similar doses of CHX have been shown previously to be effective in rats (26), and we are unaware of any data suggesting a species difference in response to CHX. Another possible mechanism would be a nonspecific interaction of CHX with COX. However, CHX has been shown not to influence activity of purified COX in vitro (12). Additionally, in the present study, the intact vasodilatory response to the nitric oxide donor SNP suggests that CHX did not have nonspecific inhibitory effects on vascular smooth muscle function. In contrast, CHX did inhibit baseline and AA-stimulated PGE₂ and prostacyclin production in brain, spleen, and muscle slices from rats. This effect of CHX and other protein synthesis inhibitors was found to be proportional to their effect on general protein synthesis inhibition (12). In this study, however, we cannot exclude the remote possibility that CHX could affect other proteins as well that may modulate COX activity. But the potent, rapid decrease in COX activity after CHX treatment may be explained by the biochemical characteristics of COX. COX is thought to be rapidly inactivated by self-produced superoxide anions, such that in an active system, COX has a half-life not more than 5-10 min (11). This suggests that maintaining active COX levels would require continuous de novo enzyme synthesis and may explain the rapid attenuation of COX-dependent vascular responses and PG synthesis after CHX administration in the piglet cerebral cortex observed in the present study. Unfortunately, immunoblotting of COX after CHX treatment would probably not yield additional support for this theory. Because COX is rapidly inactivated and degraded, functional and immunoreactive COX levels are not equivalent.

Previous evidence suggests that the PGs required for arteriolar vasodilation to hypercapnia and arterial hypotension are synthesized in the vascular endothelium (18, 20). In the present study, CHX treatment likely affected vascular endothelial cells shown by the attenuation of vascular responses to hypercapnia and arterial hypotension. We also found that 6-keto-PGF1 levels were reduced in the aCSF after CHX treatment, indicating decreased prostacyclin synthesis, further confirming the effect of CHX on cerebrovascular endothelial cells. In contrast, the reduced baseline and AA-stimulated PGF₂ levels may represent a more general inhibitory effect of CHX on COX synthesis in both neural and vascular cells.

In our previous study we demonstrated that CHX employed a dose-dependent protective effect on the NMDA-induced vasodilation (28). NMDA-induced vasodilation is a complex sequence involving the activation of neuronal NMDA receptors, activation of neuronal nitric oxide synthase (nNOS), and pial arteriolar dilation by nitric oxide (13, 22). This response was used as a bioassay to assess the functional integrity of the neuronal-vascular axis after ischemic stress. This response is attenuated by ischemia. The effect of ischemic stress likely affects the events before nNOS activation because nNOS levels and activity as well as vascular dilation to SNP were shown to be unaltered after ischemia (5). COX is an ample source of oxygen radicals in the early reperfusion period and plays a significant role in attenuating the NMDA vascular sequence after ischemia. After ischemic stress, NMDA-induced vasodilation has been shown to be preserved by pretreatment with COX inhibitors and oxygen radical scavengers, clearly indicating the involvement of COX (3-5). In our previous study, 0.3-1 mg/kg CHX was given 15 min before 10 min of global cerebral ischemia. Our present results support the concept that active COX levels could have been reduced before the initiation of ischemia. Thus the acute protective effect of CHX on the NMDA-induced vasodilation may be largely mediated via the inhibition of COX synthesis. We (23, 24) have shown similar results in cultured astroglial cells from piglets and fetal lambs.

CHX was also shown to employ long-term neuroprotective effects in different experimental ischemia models. Pretreatment with CHX was shown to reduce delayed neuronal death after transient focal ischemia (10, 21), and CHX also ameliorated cerebral infarction caused by reperfusion injury after reversible focal ischemia in rats (2). CHX has also been demonstrated to protect CA1 hippocampal neurons after transient global ischemia (14, 25). The mechanism of neuroprotection by CHX in the above-cited studies is not fully understood, but different possible mechanisms have been proposed, including CHX-induced hypothermia, inhibition of apoptosis, and suppression of the postischemic induction of a "noxious/killer protein." However, our present data reveal that pretreatment with protein synthesis inhibitors can result in not only inhibiting the appearance of a noxious/killer protein after ischemia but also in the rapid disappearance of a potentially harmful albeit continuously expressed protein: COX.

At least two distinct isoforms of COX exist (COX-1, COX-2) (9). Originally COX-1 was considered the constitutively expressed isoform, and COX-2 was designated as the inducible isoform. However, in the brain and cerebral blood vessels of newborn pigs, COX-2 but not COX-1 has been identified as the major constitutively expressed isoform (7, 27). COX-2 is an immediate early gene and is readily inducible by a wide variety of stimuli, including ischemic stress in piglet cerebral cortex and blood vessels (6, 8). There is a substantial increase in porcine cortical COX-2 mRNA levels as early as 2 h after ischemic stress, and COX-2 immunoreactivity is also increased within 8 h of cerebral ischemia. However, the short half-life and extremely rapid turnover rate of the COX enzyme may conceal an even more dramatic change in COX expression after ischemic stress. The increased expression of COX-2 may participate in the brain pathology after ischemic stress by increasing the production of oxygen radicals and inflammatory prostanoids. This is an interesting possibility, because overall protein synthesis is assumed to be inhibited even by short periods of cerebral ischemia and reperfusion (16). However, translation of some other immediate early gene mRNAs including heat shock proteins and protooncogenes appears to be increased rapidly after ischemic stress, in contrast to the generally depressed protein synthesis (15). It is quite conceivable that after cerebral ischemia, 1 mg/kg CHX has a greater inhibition on COX synthesis than we have shown with the approaches used in our present study.

In conclusion, to our knowledge we demonstrated for the first time that CHX rapidly inhibits COX activity in vivo in the cerebral cortex, as shown by the attenuation of COX-dependent pial arteriolar responses and decreased cortical metabolism of exogenous AA. This effect of CHX may be responsible for the previously reported early protective effect on neuronal and cerebrovascular function after cerebral ischemia.