Main

Intermittent and/or continuous infusions of morphine are often used for analgesia and sedation of infants in the neonatal intensive care unit. In the newborn piglet, continuous infusion of morphine results in increased systemic and sagittal sinus vein ET-1 levels (1). In vitro, ET-1 is involved in cerebral pial artery vasoconstriction, and therefore may be involved in the regulation of cerebral blood flow (2). ET-1 is a potent vasoconstrictive peptide that binds with high affinity to the endothelin receptor ETA, which mediates vasoconstriction by activating the phospholipase C/protein kinase C cascade (3), by decreasing smooth muscle sensitivity to NO (4), and possibly by increasing cytosolic free calcium levels (5) and superoxide anion production (6). The endothelin receptor ETB has a lower affinity for ET-1 and mediates vascular relaxation through a mechanism coupled to endothelial production of nitric oxide (7), although some vasoconstrictor activity of ETB has also been shown (8, 9). “Big ET” is a precursor to endothelin with almost no vasoconstrictor activity (10); ET-1 is formed when big ET-1 is cleaved by ECE (11). Published data have shown poor correlation between systemic ET levels and tissue levels of its production (12).

We have previously shown that morphine infusion in newborn piglets increases ET-1 levels, up-regulates ETA mRNA expression, and down-regulates ETB mRNA expression as measured in systemic and sagittal sinus venous blood and brainstem tissue homogenates (1). We therefore hypothesized that exposure of cultured brain vascular endothelial cells to morphine would result in increased levels of ET-1 and increased ETA mRNA expression. We further hypothesized that exposure of the same cells to the morphine antagonist naloxone would result in opposite effects. This study aimed to examine and compare the biochemical and molecular responses of the endothelin system in cultured newborn piglet brain vascular endothelial cells to morphine and naloxone in an attempt to elucidate possible cerebrovascular effects of a drug commonly used in human newborns.

METHODS

All experiments were conducted according to the guidelines of the Institutional Review Board and Institutional Animal Care and Use Committee of McGill University, Montreal, Canada. Animals were treated according to the guidelines outlined by the National Research Council Guide for the Care and Use of Laboratory Animals. Euthanasia of the animals was conducted according to the guidelines of the American Veterinary Medical Association.

Animal and cell preparation.

The brains of newborn Yorkshire piglets (1–3 d old, 1–2 kg) were perfused with 100 mL normal saline and harvested. Microvessels were extracted from the porcine brains using HBSS and Ficoll. These microvessels were plated on T25 culture flasks in endothelial cell-specific media with low serum environment (5%) containing growth factors, cytokines, and supplements (Clonetics Corporation, San Diego, CA, U.S.A.). Endothelial cells were verified by staining with factor VIII Von Willebrand factor. Cells were passaged after 3 wk (80% confluence) to T80 flasks. Cells were exposed to morphine, naloxone, or saline once cultures reached 80% confluence. Media were collected at desired time points as described in the following protocol.

Experimental protocol.

Cultured cells were divided in three groups: 1) a control group in drug-free saline media (10 mL); 2) a morphine group in 100 ng/mL morphine sulfate media (10 mL); and 3) a naloxone group in 100 ng/mL naloxone media (10 mL). Each group of cells was exposed to their respective media for time increments as follows: time 0 (n = 6), 6 h (n = 6), 24 h (n = 6), 48 h (n = 6), and 96 h (n = 6).

Extraction of ET-1 and big ET-1.

Media samples (500 μL) were collected in ice-cold polypropylene tubes containing a 500 μL solution of EDTA (1 mg/mL), and aprotinin (500 KIU/mL). The samples were extracted by addition of 1.0 mL TFA (1%) in 99% distilled water and centrifuged at 6000 ×g for 20 min at 4°C. The supernatant was loaded onto a C-18 Sep column that was previously equilibrated by washing with 100% acetonitrile followed by 1% TFA. The peptides were eluted slowly with 60% acetonitrile, 1% TFA, and 39% distilled water. The eluant was collected in a clean polypropylene tube, evaporated to dryness, and reconstituted in assay buffer.

Assay procedure.

ET-1 and big ET-1 levels were determined using enzyme immunoassay kits purchased from Peninsula Laboratories (Belmont, CA, U.S.A.). Each kit consisted of a polystyrene 96-well immunoplate precoated with peptide antibody. Aliquots of the reconstituted samples were loaded in duplicate onto the wells and the assay was carried out according to the manufacturer's protocol. Absorbance was measured at 450 nm in an automated plate reader (EAR 400 AT, SLT Labinstruments GmbH, Salzburg, Austria). The absorbance was correlated with ET-1 or big ET-1 concentrations in the standards to generate a standard curve that ranged from 0 to 1000 ng/mL. The total assay time was 4.5 h.

Total RNA isolation.

Total RNA of the sample was isolated by homogenization (1 mL/1 × 106 cells) in TriZol reagent (Invitrogen, Carlsbad, CA, U.S.A.). Chloroform (0.2 mL) was added and the tube shaken vigorously by hand. The samples were incubated for 2–3 min at room temperature then centrifuged at 14,000 rpm for 15 min at 4°C. The aqueous phase was transferred to a new tube and 0.5 mL isopropanol added. The samples were vortexed and incubated for 10 min at room temperature, then centrifuged for 10 min at 14,000 rpm at 4°C. The isopropanol was decanted and the pellet washed with 1 mL of 80% ethanol. After centrifugation for 5 min at 8000 rpm the pellet was dissolved in 50 μL of RNase free water and incubated at 55°C for 10 min in a water bath. At this point the purity of the RNA was assessed by the ratio of absorbance at 260 nm and 280 nm. The total RNA concentration was estimated by spectrophotometric measurements at 260 nm assuming 40 μg of RNA per milliliter equals one absorbance unit. The integrity of the RNA was determined by gel electrophoresis in 1% agarose gel stained with EtBr. The total RNA yield was diluted in RNase-free water to 1 μg/μL total RNA for all samples.

Reverse transcriptase (RT)-PCR.

Two micrograms of total RNA was reverse transcribed to cDNA using 100 ng random hexamers in 20 mL solution containing 50 mM Tris HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 μM each of DNTP, and 10 units MuLV reverse transcriptase (PerkinElmer Instruments, Norwalk, CT, U.S.A.) for 45 min. PCR was performed for amplification of cDNA using 1 unit AmpliTaq DNA polymerase and specific sense and anti-sense primers for β-actin, ETA, and ETB (Invitrogen). The sense and antisense primer sequences for β-actin mRNA were 5′-CCT TCC TGC GCA TGG AGT CCT GG-3′ and 5′-GGA GCA ATG ATC TTG ATC TTC-3′, respectively. The sense and antisense primer sequences for the ETA receptor mRNA were 5′-CGC TGA TAG CCA GTC TTG-3′ and 5′-CGG TTC TTG TCC ATT TCG-3′, respectively. The sense and antisense primer sequences for ETB receptor mRNA were 5′-GGA CCC ATC GAG ACT AAG G-3′ and 5′-AGA ATC CTG CTG AGG TGA AGG-3′, respectively. PCR products were amplified over 30 cycles as follows: 45 s denaturing at 94°C, 45 s primer annealing at 61°C, and 1 min extension/synthesis at 72°C.

Densitometric scanning.

Gel electrophoresis of the PCR products was performed on a 1.5% agarose gel stained with EtBr. The intensities of the bands were measured with the use of a GelDoc 1000 Darkroom Imager and Molecular Analyst software (Bio-Rad, Hercules, CA, U.S.A.). The PCR fragments were identified according to their molecular mass using a DNA mass ladder (PerkinElmer Instruments). The amount of DNA in each specimen was semi-quantitated by the integrated density of the product bands within a closed rectangle, which was then normalized to the density of the β-actin bands. Identification of the DNA fragments was accomplished with the use of a 100 base pair DNA ladder, which consisted of 15 blunt-ended fragments between 100 and 1500 base pairs. The data are expressed as mean ETA or ETB/β-actin ratio ± SEM.

Statistical analysis.

Data were analyzed using SPSS (SPSS Inc., Chicago, IL, U.S.A.) and GraphPad Prism (GraphPad Software, San Diego, CA, U.S.A.). One-way ANOVA was used to detect differences between control, morphine, and naloxone groups. Data for mRNA expression of ETA and ETB receptors are expressed as mean ETA or ETB mRNA expression over β-actin mRNA expression. Significance was determined using the Student-Newman-Keuls test, with statistical significance set at a p value of 0.05, values being expressed as mean ± SEM.

RESULTS

Effect of morphine and naloxone on ET-1 production.

Figure 1 shows the effects of morphine and naloxone on ET-1 levels. ET-1 levels for cells exposed to morphine increased from 0.52 ± 0.04 at 0 h, to 0.87 ± 0.11 at 6 h, 0.70 ± 0.08 at 24 h, 0.75 ± 0.07 at 48 h, and 0.68 ± 0.06 at 96 h (p < 0.05 at 6 h and 48 h versus saline control). In contrast, ET-1 levels (ng/mL) for cells exposed to naloxone progressively decreased from 0.51 ± 0.04 at 0 h to 0.15 ± 0.05 at 6 h, 0.25 ± 0.08 at 24 h, 0.28 ± 0.06 at 48 h, and 0.23 ± 0.03 at 96 h (p < 0.01 at 6 h and 48 h versus saline, p < 0.05 at 96 h versus saline). ET-1 levels (ng/mL) for control cells remained unchanged from 0 h (0.51 ± 0.04) through 6 h (0.58 ± 0.11), 24 h (0.43 ± 0.13), 48 h (0.55 ± 0.04), and 96 h (0.51 ± 0.08).

Figure 1
figure 1

Time course of ET-1 production by newborn piglet brain vascular endothelial cells treated with morphine sulfate (100 ng/mL media) and naloxone (100 ng/mL media). The control group was exposed to drug-free media. Data are expressed as mean ± SEM.

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Effect of morphine and naloxone on big ET-1 production.

A quite different response pattern became apparent for big ET-1 (ng/mL) production (Fig. 2). Big ET-1 levels decreased from 0.35 ± 0.04 at 0 h and 0.39 ± 0.06 at 6 h to 0.24 ± 0.15 at 24 h, 0.28 ± 0.04 at 48 h, and 0.18 ± 0.08 at 96 h (p < 0.01 at 48 h versus saline). Conversely, big ET-1 levels (ng/mL) progressively increased in response to naloxone from 0.65 ± 0.43 at 0 h, to 1.00 ± 0.09 at 6 h, 1.45 ± 0.33 at 24 h, 1.02 ± 0.10 at 48 h, and 0.97 ± 0.07 at 96 h (p < 0.05 at 24 h and 48 h versus saline, p < 0.001 at 96 h versus saline). Levels of big ET-1 in the control group were 0.31 ± 0.11 at 0 h, 0.65 ± 0.17 at 6 h, 0.71 ± 0.37 at 24 h, 0.66 ± 0.11 at 48 h, and 0.31 ± 0.12 at 96 h.

Figure 2
figure 2

Time course of big ET-1 production by newborn piglet brain vascular endothelial cells treated with morphine sulfate (100 ng/mL media) and naloxone (100 ng/mL media). The control group was exposed to drug-free media. Data are expressed as mean ± SEM.

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Effect of morphine and naloxone on ETA mRNA expression.

mRNA expression of ETA increased with morphine exposure at 6 h (2-fold, p < 0.05), 24 h (3.5-fold, p < 0.05), 48 h (2-fold, p < 0.01), and 96 h (5-fold, p < 0.01) compared with control (Fig. 3). However, compared with control, ETA mRNA expression decreased at 6 h (2.5-fold, p < 0.05), 24 h (2-fold), 48 h (11-fold, p < 0.01), and 96 h (17-fold, p < 0.05) in the naxolone-exposed cells.

Figure 3
figure 3

Response of ETA receptor to morphine and naloxone in newborn piglet brain vascular endothelial cells. The graph represents the mean ratio ± SEM of each ETA mRNA to β-actin mRNA expression. Samples from six flasks in each group were analyzed.

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Effect of morphine and naloxone on ETB mRNA expression.

Figure 4 illustrates the mRNA expression of ETB in response to morphine and naloxone. Morphine treatment had no significant effect on mRNA expression of ETB by newborn piglet brain vascular endothelial cells. In contrast, naloxone exposure resulted in a 3-fold increase in ETB mRNA expression at 48 and 96 h (p < 0.001) compared with control levels.

Figure 4
figure 4

Response of ETB receptor to morphine and naloxone in newborn piglet brain vascular endothelial cells. The graph represents the mean ratio ± SEM of each ETB mRNA to β-actin mRNA expression. Samples from six flasks in each group were analyzed.

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Figure 5 represents gels from a single sample taken from a group of six samples at each time interval, for RT-PCR amplification of newborn piglet brain vascular endothelial cell β-actin, ETA and ETB receptor mRNA expression. Lanes 1–6 represents 0–96 h of incubation, respectively. This figure demonstrates that morphine increases ETA and decreases ETB mRNA in newborn piglet brain vascular endothelial cells, whereas an opposite effect is noted with naloxone.

Figure 5
figure 5

This figure represents gels of a single sample taken from a group of six samples at each time interval, for RT-PCR amplification of newborn piglet brain vascular endothelial cell β-actin, ETA,and ETB receptor mRNA expression. Lane 1 = 0 h; lane 2 = 6 h; lane 3 = 24 h; lane 4 = 48 h; and lane 5 = 96 h of incubation.

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DISCUSSION

Although no significant change in ET-1, big ET-1, ETA mRNA, or ETB mRNA were noted with saline-exposed controls of brain vascular endothelial cells, the first test of our hypothesis is demonstrated by the effects of morphine on ET-1 production in the media. Morphine exposure led to increased levels of ET-1 at 6 and 48 h, decreased levels of big ET-1 at 48 h, and increased ETA receptor mRNA at 6, 24, 48, and 96 h, with no appreciative effect on ETB receptor mRNA expression. These responses of brain vascular endothelial cells to morphine exposure would be expected to lead to vasoconstriction of cerebral vasculature in vivo(3, 6, 13, 14) as well as to have a mitogenic effect on vascular smooth muscle (11, 13–15). Other investigators have noted hypotensive effects of opioids, possibly due to stimulation of NO release by Mu-3 receptor activation (16). This opioid-induced vasodilation in newborn piglets has been found to be partially restored after blockade of ET-1 receptors (6). A recent study by Hu et al.(17) demonstrated that morphine induces apoptosis in primary human fetal microglial and neuronal cell cultures, an effect that was blocked by naloxone. Thus, the present findings are consonant with previous studies. Elevation of ET-1 levels with reduction of big ET-1 levels may indicate increased activity of ECE, as opposed to increased production of ET precursors (prepro ET or big ET) (18). Morphine has been shown to increase activity of angiotensin converting enzyme (ACE), presumably by interactions within the nucleus of endothelial cells and not mediated by opioid receptors (18). Effects of morphine on ET receptor expression leading to changes in ET and/or big ET levels via feedback mechanisms is also possible (19, 20), although less likely given the effects on ETA and ETB receptor mRNA expression.

The second test of our hypothesis that naloxone exposure would result in responses opposite to that of morphine is demonstrated in Figures 1 through 5. Naloxone exposure led to significantly decreased levels of ET-1 at 6, 48, and 96 h; increased levels of big ET-1 at 24, 48, and 96 h; decreased ETA receptor mRNA at 6, 48, and 96 h; and increased ETB receptor mRNA at 48 and 96 h. Interestingly, these effects of naloxone on the piglet brain vascular endothelial cells were the result of primary exposure to naloxone, rather than the result of naloxone rescue of cells previously exposed to morphine. Naloxone is a competitive antagonist of opioids thought to have essentially no pharmacologic activity in the absence of administered or endogenous opioid agonists (21, 22). Our findings of essentially opposite effects of naloxone on endothelin-related responses compared with morphine suggest the effects of morphine on ET-1 and its receptors are mediated by opiate receptors potentially blocked by naloxone. Activity of naloxone in the absence of prior exposure of the cell culture to morphine implies the presence of endogenous opioids in the piglet brain vascular endothelial cells. Our findings that opioid receptor antagonism increased levels of big ET-1 while decreasing levels of ET-1 again lend evidence to the existence of opioid receptors with influence upon ECE activity. The dose of morphine used in the present study was based on pharmacokinetic studies using single morphine doses of 0.1 mg/kg in preterm infants (23) and loading doses of 0.1 mg/kg/h for 2 h, followed by infusion doses (24), which demonstrated serum levels of 100 ng/mL or greater. One limitation of the study is that we did not measure the levels of morphine or its metabolites in the media. Furthermore, pretreatment of the cells with naloxone, followed by morphine exposure would have offered other possible explanations to our findings.

CONCLUSIONS

In summary, morphine and naloxone exposure of cultured newborn piglet brain vascular endothelial cells led to changes in levels of ET-1 and expression of its receptors. The changes noted would be expected to induce brain vascular constriction and changes in cerebral blood pressure and blood flow. In preterm infants such changes in cerebral blood flow could be a risk factor for development of IVH (25) or PVL (26), important morbidities in the preterm newborn population. Recent findings of the Neopain Multicenter Trial Group demonstrated that the combination of death, IVH, and PVL occurred more frequently in infants of gestational ages between 27 and 29 wk who were treated with 0.1 mg/kg morphine loading dose followed by infusions of 20 μg/kg/h (27). Further investigation of the effects of opioid administration on human newborn infants will be necessary to more precisely identify any true risks of such drug administration. Contributions of the various ET receptors to vasoconstriction seem to vary greatly among species, and among anatomic locations within species (12), and effects of morphine may differ in anesthetized versus unanesthetized subjects (28). Also, preterm human infants may be somewhat protected from the vasoactive effects of ET-1 due to lack of development of the muscularis layer in some cerebral arteries (29). Interactions between opioids and ET in vivo are likely very complex, possibly including regulatory mechanisms such as induction of eventual hydrolytic inactivation of ET-1 via endopeptidases by morphine infusion (30). We are currently undertaking a study on human preterm infants at our institution to evaluate effects of opioid administration on levels of ET and other vasoactive mediators, in an effort to better understand the possibly harmful effects of a routinely prescribed medication for preterm infants.